Involvement of protein kinase C-alpha and -epsilon in extracellular Ca signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca²⁺ concentration ([Ca²⁺]_o) and modulation of cellular processes associated with acute or sustained changes in [Ca²⁺]_o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca²⁺]_o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca²⁺]_o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca²⁺]_o required influx of Ca²⁺through Ni²⁺-sensitive Ca²⁺channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca²⁺]_o. Activation of ERK1/2 by high [Ca²⁺]_o was not necessary for the [Ca²⁺]_o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca²⁺]_o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Transepithelial transport of flavanone in intestinal Caco-2 cell monolayers

Our recent study [S. Kobayashi, S. Tanabe, M. Sugiyama, Y. Konishi, Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers, Biochim. Biophys. Acta, 1778 (2008) 33-41] shows that the mechanism of absorption of hesperetin involves both proton-coupled active transport and transcellular passive diffusion. Here, as well as analyzing the cell permeability of hesperetin, we also study the transport of other flavanones, naringenin and eriodictyol, using Caco-2 cell monolayers. Similar to hesperetin mentioned, naringenin and eriodictyol showed proton-coupled polarized transport in apical-to-basolateral direction in non-saturable manner, constant permeation in the apical-to-basolateral direction (J_ap → bl) irrespective of the transepithelial electrical resistance (TER), and preferable distribution into the basolateral side after apical loading in the presence of a proton gradient. Furthermore, the proton-coupled J_ap → bl of hesperetin, naringenin and eriodictyol, were inhibited by substrates of the monocarboxylic acid transporter (MCT), such as benzoic acid, but not by ferulic acid. In contrast, both benzoic and ferulic acids have no stimulatory effect on J_ap → bl of each flavanone by trans-stimulation analysis. These results indicates that proton-driven active transport is commonly participated in the absorption of flavanone in general, and that its transport is presumed to be unique other than MCT-mediated transport for absorption of phenolic acids (PAs), sodium-dependent MCT (SMCT) nor anion exchanger-mediated transport.     

Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC₅₀ measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin''s cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.     

Activation of choline kinase by extracellular Ca is Ca-sensing receptor, Gα12 and Rho-dependent in breast cancer cells

Breast cancer cell metastases to bone result in osteolysis and release of large quantities of Ca²⁺ into the bone microenviroment. Extracellular Ca²⁺ (Ca_o²⁺) acting through the Ca²⁺-sensing receptor (CaR), a member of G protein-coupled receptor superfamily, plays an important role in the regulation of multiple signaling pathways. Here, we find that expression of the CaR and Gα₁₂ is significantly up-regulated in breast cancer cells (MDA-MB-231 and MCF-7) compared with nonmalignant breast cells (Hs 578Bst and MCF-10A). Ca_o²⁺ induces a significant increase in extracellular [³H]phosphocholine (P-cho) production in breast cancer cells. Using an anti-CaR antibody to block Ca_o²⁺ binding to the CaR and small interfering RNA (siRNA) to silence CaR gene expression, our data demonstrate that [³H]P-cho production in response to Ca_o²⁺-stimulation is CaR-dependent. By analyzing cellular lipid profiles and using siRNA to silence choline kinase (ChoK) expression, we determine that the production of [³H]P-cho is primarily related to CaR-induced ChoK activation, and not degradation of choline phospholipids. Finally, by pretreatment of the cells with either pertussis toxin or C₃ exoenzyme, co-immunoprecipiation of Gα_i, Gα_q or Gα₁₂ with the CaR, and RhoA translocation, we found that the enhancement of ChoK activation and P-cho production in breast cancer cells occurs via a CaR-Gα₁₂-Rho signaling pathway.     

Small Synthetic Peptides Homologous to Segments of the First External Loop of Occludin Impair Tight Junction Resealing

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca²⁺ removal and was characterized by a marked drop of TER. The reintroduction of Ca²⁺ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca²⁺ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions. Received: 4 December 1998/Revised: 22 January 1999     

Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca²⁺ concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.     

Activation of the calcium-sensing receptor by high calcium induced breast cancer cell proliferation and TRPC1 cation channel over-expression potentially through EGFR pathways

The calcium sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium ([Ca²⁺]_o). In MCF-7 human breast cancer cells, we previously reported that treatment with [Ca²⁺]_o for 24 h leads to an over-expression of the Transient Receptor Potential Canonical 1 (TRPC1) cation channel and cell proliferation. Both involve the extracellular signal-regulated Kinases 1 & 2 (ERK1/2). MCF-7 also expressed epidermal growth factor receptor (EGFR) which is involved in cell proliferation through ERK1/2. Therefore, we investigated the cross-talk between CaR and EGFR in mediating ERK1/2 phosphorylation, TRPC1 over-expression and cell proliferation. Our data show that both high [Ca²⁺]_o and EGF phosphorylate ERK1/2. Furthermore, inhibition of EGFR kinase and matrix metalloproteinases (MMPs) reduced the overall effects mediated by [Ca²⁺]_o such as activation of ERK1/2, expression of TRPC1 and cell proliferation. They indicate the important role of the CaR-EGFR-ERK axis in transmitting mitogenic signals generated by high [Ca²⁺]_o in MCF-7 cells.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

Action of mercurials on the active and passive transport properties of sarcoplasmic reticulum

The effect of Hg²⁺ and Ch₃-Hg⁺ on the passive and active transport properties of the Ca²⁺-Mg²⁺-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10^–5 and 10^–4 M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca²⁺. The mercurials increase the passive Ca²⁺ and Mg²⁺ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca²⁺-Mg²⁺-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K⁺ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the Ca²⁺-Mg²⁺-ATPase and other SR proteins in black lipid membranes, as presented by others.     

Cortisol stimulates calcium transport across cultured gill epithelia from freshwater rainbow trout

Summary The effect of cortisol on calcium (Ca²⁺) transport across cultured rainbow trout gill epithelia composed of both pavement cells (PVCs) and mitochondria-rich cells (MRCs) was examined. Under symmetrical culture conditions (L15 media apical/L15 media basolateral), cortisol had subtle effects on gill epithelial preparations. Both control and cortisol treated epithelia exhibited Ca²⁺ influx and efflux rates (measured radioisotopically using ⁴⁵Ca) that were approximately balanced, with a slight inwardly directed net Ca²⁺ flux. Ussing flux ratio analysis indicated active Ca²⁺ transport in the inward direction across epithelia bathed symmetrically regardless of hormone treatment. In contrast, under asymmetrical conditions (freshwater apical/L15 media basolateral) control epithelia exhibited active Ca²⁺ transport in the outward direction (basolateral to apical) throughout experiments conducted over a 24-h period, whereas cortisol-treated preparations exhibited active transport in the inward direction (apical to basolateral) during the early stages of an asymmetrical culture period (e.g., T0–6 h) and passive transport during the later stages (e.g., T18–24 h). When soft freshwater (with tenfold lower [Ca²⁺]) was used for asymmetrical culture instead of freshwater, control epithelia developed outwardly directed active Ca²⁺ transport properties, whereas cortisol-treated preparations did not. The results of this study support a hypercalcemic role for cortisol in rainbow trout and demonstrate that treating cultured gill epithelia composed of both PVCs and MRCs with cortisol can stimulate active Ca²⁺ uptake under circumstances that more closely resemble natural conditions for fish gills (i.e., freshwater bathing the apical side of the epithelium).     

G protein-coupled, extracellular Ca2+ (Ca o 2+ )-sensing receptor enables Ca o 2+ to function as a versatile extracellular first messenger

The cloning of a G protein-coupled, extracellular Ca²⁺ (Ca _o ²⁺ )-sensing receptor (CaR) has afforded a molecular basis for a number of the known effects of Ca _o ²⁺ on tissues involved in maintaining systemic calcium homeostasis, especially parathyroid gland and kidney. In addition to providing molecular tools for showing that CaR messenger RNA and protein are present within these tissues, the cloned CaR has permitted documentation that several human diseases are the result of inactivating or activating mutations of this receptor as well as generation of mice that have targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in these patients as well as in the CaR “knockout” mice have elucidated considerably the CaR’s physiological roles in mineral ion homeostasis. Nevertheless, a great deal remains to be learned about how this receptor regulates the functioning of other tissues involved in Ca _o ²⁺ metabolism, such as bone and intestine. Further study of these human diseases and of the mouse models will doubtless be useful in gaining additional understanding of the CaR’s roles in these latter tissues. Furthermore, we understand little of the CaR’s functions in tissues that are not directly involved in systemic mineral ion metabolism, where the receptor probably serves diverse other roles. Some of these functions may be related to the control of intra- and local extracellular concentrations of Ca²⁺, while others may be unrelated to either systemic or local ionic homeostasis. In any case, the CaR and conceivably additional receptors/sensors for Ca²⁺ or other extracellular ions represent versatile regulators of a wide variety of cellular functions and represent important targets for novel classes of therapeutics.     

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5

The extracellular Ca²⁺-sensing receptor (CaR) is a key-player in plasma Ca²⁺ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca²⁺ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca²⁺-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca²⁺ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca²⁺ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR^R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca²⁺ influx via a PMA-insensitive PKC isoform pathway.     

Effects of capsaicin on human intestinal cell line Caco-2

The influence of capsaicin processing on human intestinal cell line Caco-2 was examined by measuring transepithelial electrical resistance (TER). There was an increase in permeability at high concentration (200 to 500 μM) of capsaicin, and the effect was inhibited by pretreatment of capsazepine, which is a competitive antagonist of the vanilloid receptor 1 (VR1). LDH-activity as well as changes in intracellular Ca²⁺ were determined to know whether or not capsaicin affected TER activity through its influence on the tight junction. We also determined the expression of the VR1-like protein on Caco-2 cells in time-dependent manner by western blotting using vanilloid receptor (VR1) antiserum. Our results showed that the permeability increase by capsaicin was through binding to VR1-like protein of Caco-2 cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm² with 50 μM PC, and 17.0 and 23.0 nmol/h/cm² with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)     

Magnesium–calcium signalling in rat parotid acinar cells: effects of acetylcholine

This study investigated the effects of extracellular Mg²⁺ ([Mg²⁺]_o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca²⁺ ([Ca²⁺]_i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg²⁺]_o, ACh evoked significant increases in amylase secretion and [Ca²⁺]_i. Either low (0 mM) or elevated (5 and 10 mM) [Mg²⁺]_o attenuated ACh-evoked responses. In a nominally Ca²⁺ free medium, elevated [Mg²⁺]_o attenuated basal and ACh-evoked amylase secretion and [Ca²⁺]_i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg²⁺]_o, ACh evoked a gradual decrease in [Mg²⁺]_i. These results indicate that the ACh-evoked Mg²⁺ efflux is an active process since Mg²⁺ has to move against its gradient. Either lidocaine, amiloride, N-methyl-d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg²⁺]_i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg²⁺]_i but the response was less pronounced with either [Na⁺]_o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca²⁺ and Mg²⁺ signalling in parotid acinar cells and that ACh-evoked Mg²⁺ transport was not dependent upon [Na⁺]_o.     

The role of calcium ions in the maintenance of resting potentials and ionic gradients of mollusk neurons

We studied on apple snail neurons the connection between K⁺ and Na⁺ concentration gradients, transmembrane difference of potentials, and concentrations of Ca²⁺ in the external medium. Sensitivity of the resting potential (RP) of neurons to the influence of temperature and to metabolic poisons rose considerably with a decrease of Ca²⁺ concentration in the solution surrounding a ganglion. An excess of Ca²⁺ in the external medium did not affect the RP or ion concentration in nerve cells. Removal of Na²⁺ from this solution causes hyperpolarization of the membrane which disappears when active transport of sodium ions through the membrane is suppressed. Sodium enrichment and potassium impoverishment of the neurons are observed in potassium-free solutions at 4°C. Reaccumulation of K⁺ and exclusion of Na⁺ from the solutions of 21°C depends on the concentration of Ca²⁺ in the medium. The ionic composition of the neurons is not restored upon removal of Ca²⁺ from the solution. Upon increasing the amount of Ca²⁺, movement of ions against the concentration gradients is intensified. Thus, it may be concluded that Ca²⁺ ions on the one hand participate in the maintenance of normal passive permeability of ions through the membrane, and on the other accelerate active transport of K⁺ and Na⁺ against the concentration gradients. The mechanisms of these processes are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 323–330, November–December, 1969.     

Methylated N-(4-N,N-Dimethylaminobenzyl) Chitosan, a Novel Chitosan Derivative, Enhances Paracellular Permeability Across Intestinal Epithelial Cells (Caco-2)

The aim of this study was to investigate the effect of methylated N-(4-N,N-dimethylaminobenzyl) chitosan, TM-Bz-CS, on the paracellular permeability of Caco-2 cell monolayers and its toxicity towards the cell lines. The factors affecting epithelial permeability, e.g., degree of quaternization (DQ) and extent of dimethylaminobenzyl substitution (ES), were evaluated in intestinal cell monolayers of Caco-2 cells using the transepithelial electrical resistance and permeability of Caco-2 cell monolayers, with fluorescein isothiocyanate dextran 4,400 (FD-4) as a model compound for paracellular tight-junction transport. Cytotoxicity was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide viability assay. The results revealed that, at pH 7.4, TM-Bz-CS appeared to increase cell permeability in a concentration-dependent manner, and this effect was relatively reversible at lower doses of 0.05–0.5 mM. Higher DQ and the ES caused the permeability of FD-4 to be higher. The cytotoxicity of TM-Bz-CS depended on concentration, %DQ, and %ES. These studies demonstrated that this novel modified chitosan has potential as an absorption enhancer.     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

The effect of calcium ion transport ATPase upon the passive calcium ion permeability of phospholipid vesicles

The uptake and release of Ca²⁺ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca²⁺ indicator.Incorporation of the Ca²⁺ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca²⁺ by several orders of magnitude. Therefore in addition to participating in active Ca²⁺ transport, the ($\text{Mg}{}^{\mathrm{2+}}\text{+ Ca}{}^{\mathrm{2+}}$)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca²⁺ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca²⁺ release from the sarcoplasmic reticulum during activation of muscle.