Preparation of casein phosphopeptides calcium complex and the promotion in calcium cellular uptake through transcellular transport pathway

Vitamin C (VC)—a collective term for the different oxidation and protonation forms of ascorbic acid (AscH)—is an essential micronutrient that serves as (i) a potent antioxidant and (ii) a cofactor of a manifold of enzymatic processes. Its role in health is related to redox balance maintenance, which is altered in diseases such as obesity, cancer, neurodegenerative diseases, hypertension, and autoimmune diseases. Despite its importance, VC uptake has been poorly investigated. Available literature values for the passive membrane permeability P of lipid bilayers for AscH scatter by about 10 orders of magnitude. Here, we show by voltage clamp that P − of AscH’s anionic form (ascorbate Asc − ) is negligible. To cross the membrane, Asc − picks up a proton in the membrane vicinity and releases it on the other side of the membrane. This leads to a near-membrane pH drop that was visualized by scanning pH microelectrodes. The AscH concentration dependent pH profiles indicated P = 1.1 ± 0.1 × 10 − 8 cm / s . Thus, AscH’s P is comparable to that of sorbitol and much lower than that of other weak acids like acetic acid or salicylic acid. The observation suggests that the capacity of the passive transcellular transport pathway across the lipid matrix does not suffice to ensure the required VC intake from the gastrointestinal tract.

Download Full-text

NANO-BIOTECHNOLOGY AND ITS INNOVATIVE PERSPECTIVE IN DIABETES MANAGEMENT

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557521666210623164052 ◽

2021 ◽

Vol 21 ◽

Author(s):

Jigar Raval ◽

Riddhi Trivedi ◽

Sonali Suman ◽

Arvind Kukrety ◽

Prajesh Prajapati

Keyword(s):

Cellular Uptake ◽

Glucose Homeostasis ◽

Diabetes Management ◽

The Body ◽

Paracellular Transport ◽

Specific Gene ◽

Future Prospects ◽

Transcellular Transport ◽

Routes Of Administration ◽

Two Stages

: Diabetes occurs due to the imbalance of glucose in the body known as glucose homeostasis, thus leading to metabolic changes in the body. The two stages hypoglycemia or hyperglycemia classify diabetes into various categories. Various bio-nanotechnological approaches are coupled up with nano particulates, polymers, liposome, various gold plated and solid lipid particulates, regulating transcellular transport, non specific cellular uptake, and paracellular transport, leading to oral, trans-dermal , pulmonary, buccal , nasal , specific gene oriented administration to avoid the patient’s non compliance with the parental routes of administration. Phytochemicals are emerging strategies for the future prospects of diabetes management.

Download Full-text

X-ray microanalysis of intracellular ions in epithelial tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100132534 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1580-1581

Author(s):

Roger Rick

Keyword(s):

Cell Types ◽

Quantitative Detection ◽

Transport Pathway ◽

Transcellular Transport ◽

X Ray ◽

Epithelial Tissues ◽

Nominal Thickness ◽

Intracellular Compartments ◽

The Moment ◽

Different Cell Types

Electron probe x-ray microanalysis provides a powerful tool in studying transepithelial ion transport mechanisms as it allows the quantitative detection of diffusible ions on a cellular and subcellular scale. The high spatial resolution afforded by this method is of particular advantage in epithelial tissues that are composed of different cell types and cases in which the transcellular transport pathway might involve different intracellular compartments. In the following, the methods applied in several studies are described, with emphasis on some recent modifications.The tissues are frozen by rapid immersion in liquified ethane kept at -180°C. Support systems for different epithelial preparations have been developed to facilitate freezing and maintain the functional state of the epithelium immediately up to the moment of freezing. A fluid-clamp technique is used to mount the tissue for sectioning (Figure 1). Sections are cut dry at -140°C and a nominal thickness of 200 nm using 40° glass knifes. The sections are picked up from the back of the knife by a fire-polished wire probe (1 μm tip diameter) and placed on a parlodion film which is suspended over a 2 mm opening in a special beryllium carrier disc.

Download Full-text

Movement of Hydroxyurea Across Cell Membranes Is Mediated by Specific Solute Carrier (SLC) Transporters.

Blood ◽

10.1182/blood.v114.22.2575.2575 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2575-2575

Author(s):

Aisha L. Walker ◽

Ryan M Franke ◽

Alex Sparreboom ◽

Russell E. Ware

Keyword(s):

Vector Control ◽

Cellular Uptake ◽

Organic Cation ◽

Organic Anion ◽

Fold Increase ◽

The Body ◽

Transcellular Transport ◽

Slc Transporters ◽

Solute Carrier

Abstract Abstract 2575 Poster Board II-552 Background: Hydroxyurea is the only FDA-approved drug for the treatment of sickle cell anemia (SCA) in adults. Hydroxyurea increases fetal hemoglobin, decreases hospitalizations and painful events, and reduces mortality. With an oral bioavailability of > 90%, hydroxyurea is rapidly absorbed and distributed throughout the body. Though hydroxyurea has proven to be effective in treating SCA, there is considerable inter-patient variability observed in the pharmacokinetics and pharmacodynamics of hydroxyurea. Currently, mechanisms involved in the absorption, distribution, and elimination of hydroxyurea remain unclear. Recently, key transmembrane proteins have been identified as drug transporters due to their ability to move a variety of xenobiotic substances across cell membranes. Drug transporters are widely distributed throughout the body, and most are specific to certain substrates. Solute carrier (SLC) transporters in particular have been to shown to significantly impact drug pharmacokinetics by influencing the absorption, distribution, and elimination of specific drugs. The present study was designed to identify SLC transporters that may influence the absorption, distribution, and/or elimination of hydroxyurea in patients with SCA. Methods: In vitro studies using an equilibrium dialysis plate were performed to determine the amount of hydroxyurea that binds to human serum proteins. Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [14C]-hydroxyurea accumulation in HEK293 cells and oocytes that overexpress organic anion transporters (OAT1-3), organic cation transporters (OCT1-3), organic cation/carnitine transporters (OCTN1-2), organic anion transporting polypeptides (OATP1A2/OATP1B1/OATP1B3), or vector control. LLC-PK1 cells that overexpress urea transporters A or B (UTA/UTB) were used to determine UTA/UTB mediated transcellular transport of hydroxyurea in transwell plates. The transport of [14C]-hydroxyurea from apical to basal or from basal to apical compartments was measured for the UTA/UTB overexpressing cells and compared to vector control. UTA and UTB mRNA expression was measured by real-time PCR of cDNA obtained from human tissue samples. Results: Protein binding assays showed that >76% of [14C]-hydroxyurea remained unbound to proteins in human serum containing hydroxyurea at concentrations ranging from 1.5μM to 500μM. The fraction of unbound hydroxyurea was similar using serum obtained from pediatric patients with SCA. In uptake studies, [14C]-hydroxyurea was a potent substrate for OATP1B3 with an approximately 2-fold increase in drug accumulation compared to control (p<0.001). In contrast, hydroxyurea was found to be a weak substrate for OCTN1, OCTN2, OATP1A2, and OATP1B1 with only a 1.3-fold increase in drug accumulation compared to control (p<0.04). Transcellular transport of hydroxyurea was increased 3- and 2-fold by UTA and UTB, respectively, compared to vector control demonstrating hydroxyurea to be a potent substrate for these transporters as well (p<0.02). When the urea transporter inhibitor dimethylurea was added, hydroxyurea transport by UTA and UTB-expressing cells was decreased to levels observed with the vector control. In real-time PCR assays, kidney, muscle, and small intestine were among human tissues with high expression of UTA mRNA, while prostate, brain, and bone marrow had high levels of UTB mRNA expression. Conclusion: Cellular uptake of hydroxyurea is mediated by active transport via specific SLC transporters OATP1B3, UTA and UTB, which are expressed in liver, kidney, brain, intestine, and blood cells. Studies to further characterize hydroxyurea transporters should improve our understanding of the pharmacokinetic and pharmacodynamic profiles of hydroxyurea used in clinical practice for patients with SCA. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2113 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2113-2123 ◽

Cited By ~ 89

Author(s):

C A Hoppe ◽

T P Connolly ◽

A L Hubbard

Keyword(s):

Morphological Characterization ◽

Intact Protein ◽

Transport Pathway ◽

Transcellular Transport ◽

Peroxidase Cytochemistry ◽

Parenchymal Cells ◽

Intracellular Pathway ◽

Mouse Myeloma

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.

Download Full-text

Paracellular calcium transport across renal and intestinal epithelia

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0061 ◽

2014 ◽

Vol 92 (6) ◽

pp. 467-480 ◽

Cited By ~ 34

Author(s):

R. Todd Alexander ◽

Juraj Rievaj ◽

Henrik Dimke

Keyword(s):

Thick Ascending Limb ◽

Transport Pathway ◽

Transport Pathways ◽

Transcellular Transport ◽

Renal Epithelium ◽

Intestinal Epithelia ◽

Physiological Processes ◽

Renal Tubular Reabsorption ◽

Renal Tubular ◽

Paracellular Shunt

Calcium (Ca2+) is a key constituent in a myriad of physiological processes from intracellular signalling to the mineralization of bone. As a consequence, Ca2+ is maintained within narrow limits when circulating in plasma. This is accomplished via regulated interplay between intestinal absorption, renal tubular reabsorption, and exchange with bone. Many studies have focused on the highly regulated active transcellular transport pathways for Ca2+ from the duodenum of the intestine and the distal nephron of the kidney. However, comparatively little work has examined the molecular constituents creating the paracellular shunt across intestinal and renal epithelium, the transport pathway responsible for the majority of transepithelial Ca2+ flux. More specifically, passive paracellular Ca2+ absorption occurs across the majority of the intestine in addition to the renal proximal tubule and thick ascending limb of Henle’s loop. Importantly, recent studies demonstrated that Ca2+ transport through the paracellular shunt is significantly regulated. Therefore, we have summarized the evidence for different modes of paracellular Ca2+ flux across renal and intestinal epithelia and highlighted recent molecular insights into both the mechanism of secondarily active paracellular Ca2+ movement and the identity of claudins that permit the passage of Ca2+ through the tight junction of these epithelia.

Download Full-text

The Glycocalyx and Pressure-Dependent Transcellular Albumin Transport

Cardiovascular Engineering and Technology ◽

10.1007/s13239-020-00489-5 ◽

2020 ◽

Vol 11 (6) ◽

pp. 655-662 ◽

Cited By ~ 1

Author(s):

Randal O. Dull ◽

Andreia Z. Chignalia

Keyword(s):

Endothelial Cells ◽

Hydrostatic Pressure ◽

Solute Flux ◽

Microvascular Endothelial Cells ◽

Rat Lung ◽

Transport Pathway ◽

Transcellular Transport ◽

Microvascular Endothelial ◽

Heparan Sulfates ◽

Uptake And Transport

Abstract Purpose Acute increases in hydrostatic pressure activate endothelial signaling pathways that modulate barrier function and vascular permeability. We investigated the role the glycocalyx and established mechanotransduction pathways in pressure-induced albumin transport across rat lung microvascular endothelial cells. Methods Rat lung microvascular endothelial cells (RLMEC) were cultured on Costar Snapwell chambers. Cell morphology was assessed using silver nitrate staining. RLMEC were exposed to zero pressure (Control) or 30 cmH2O (Pressure) for 30 or 60 min. Intracellular albumin uptake and transcellular albumin transport was quantified. Transcellular transport was reported as solute flux (Js) and an effective permeability coefficient (Pe). The removal of cell surface heparan sulfates (heparinase), inhibition of NOS (L-NAME) and reactive oxygen species (apocynin, Apo) was investigated. Results Acute increase in hydrostatic pressure augmented albumin uptake by 30–40% at 60 min and Js and Pe both increased significantly. Heparinase increased albumin uptake but attenuated transcellular transport while L-NAME attenuated both pressure-dependent albumin uptake and transport. Apo interrupted albumin uptake under both control and pressure conditions, leading to a near total lack of transcellular transport, suggesting a different mechanism and/or site of action. Conclusion Pressure-dependent albumin uptake and transcellular transport is another component of endothelial mechanotransduction and associated regulation of solute flux. This novel albumin uptake and transport pathway is regulated by heparan sulfates and eNOS. Albumin uptake is sensitive to ROS. The physiological and clinical implications of this albumin transport are discussed.

Download Full-text

A novel transcellular transport pathway for non-bile salt cholephilic organic anions

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1991.261.2.g305 ◽

1991 ◽

Vol 261 (2) ◽

pp. G305-G311 ◽

Cited By ~ 1

Author(s):

N. Aoyama ◽

H. Tokumo ◽

T. Ohya ◽

K. Chandler ◽

R. T. Holzbach

Keyword(s):

Bile Salt ◽

Inhibitory Effect ◽

Organic Anions ◽

Isolated Perfused Rat Liver ◽

Phenol Red ◽

Transport Pathway ◽

Single Class ◽

Transcellular Transport ◽

Tracer Dose ◽

Saturation Dose

Non-bile salt cholephilic organic anions comprise a single class of nonhomologous ligands having a range of hydrophobicity. Hydrophobicity enhances the hepatic extraction of cholephiles as well as their partitioning into secreted biliary lipid particles. When hydrophobicity is correlated with patterns of biliary excretion for studying transcellular transport, however, the more hydrophobic probes are unsuitable. Specifically, with the isolated perfused rat liver technique, the excretory times for sulfobromophthalein and rose bengal were significantly longer compared with that for the much more hydrophilic analogue phenol red (PR), which showed only a single, nearly symmetrical excretory peak at 10 min. Colchicine affected the apparently well-defined PR pathway only at a saturation dose (10,000 times the tracer dose). In contrast, the effect of a different perturbant, monensin, was striking at a tracer dose of PR, but was less evident at a saturation dose. The combined administration of colchicine and monensin had no additive inhibitory effect on PR excretion at tracer doses. At a saturation dose of PR, where monensin is less inhibiting, however, a significant additive inhibitory effect was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

Water-soluble amphiphilic ruthenium(ii) polypyridyl complexes as potential light-activated therapeutic agents

Chemical Communications ◽

10.1039/d0cc04397d ◽

2020 ◽

Vol 56 (65) ◽

pp. 9332-9335

Author(s):

Sandra Estalayo-Adrián ◽

Salvador Blasco ◽

Sandra A. Bright ◽

Gavin J. McManus ◽

Guillermo Orellana ◽

...

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Cellular Uptake ◽

Therapeutic Agents ◽

Water Soluble ◽

Polypyridyl Complexes ◽

Cervical Cancer Cells

Two new water-soluble amphiphilic Ru(ii) polypyridyl complexes were synthesised and their photophysical and photobiological properties evaluated; both complexes showed a rapid cellular uptake and phototoxicity against HeLa cervical cancer cells.

Download Full-text