Viscoelasticity of basal plasma membranes and cortices derived from MDCK II cells

In mature epithelial cells, however, cells adhere to one another through tight junctions, adherens junctions and desmosomes thereby displaying a pronounced apical-basal polarity. In vivo, the apical membrane has a larger surface area and faces the outer surface of the body or the lumen of internal cavities, whereas the basolateral membrane is oriented on the side away from the lumen and forms focal adhesions with the extracellular matrix. The mechanical properties of cells are largely determined by the architecture and dynamics of their viscoelastic cortex, which consists of a contractile, cross-linked actin mesh attached to the plasma membrane via linker proteins. Measuring the mechanical properties of adherent, polarized epithelial cells is usually limited to the upper, i.e., apical side of the cells due to their accessibility on culture dishes. Moreover, contributions from the cell interior comprising various filament types, organelles, and the crowded cytoplasm usually impede examination of the cortex alone. Here, we investigate the viscoelastic properties of basolateral membranes derived from polarized MDCK II epithelia in response to external deformation and compare them to living cells probed at the apical side. Therefore, we grew MDCK II cells on porous surfaces to confluency and removed the upper cell body by sandwich cleavage. The free-standing, defoliated cortices were subject to force indentation and relaxation experiments permitting a precise assessment of cortical viscoelasticity. A new theoretical framework to describe the force cycles is developed and applied to obtain the time-dependent area compressibility modulus of cell cortices from adherent cells. Compared to the viscoelastic response of living cells the basolateral membranes are substantially less fluid and stiffer but obey to the same universal scaling law if excess area is taken into account.

Parallel intermediate conductance K+ and Cl− channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon

This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller but significant contribution from K+-Cl− cotransporter-1 activity.

Putative linear motifs mediate the trafficking to apical and basolateral membranes

Cell polarity refers to the asymmetric organisation of cellular components in various cells. Epithelial cells are the best known examples of polarized cells, featuring apical and basolateral membrane domains. Despite huge efforts, the exact rules governing the protein distribution in such domains are still elusive. In this study we examined linear motifs accumulating in these parts and based on the results we prepared ‘Classical’ and Convolutional Neural Networks to classify human transmembrane proteins localizing into apical/basolateral membranes. Asymmetric expression of drug transporters results in vectorial drug transport, governing the pharmacokinetics of numerous substances, yet the data on how proteins are sorted in epithelial cells is very scattered. The provided dataset may offer help to experimentalists to characterize novel molecular targets to regulate transport processes more precisely.

Structural proteins of plasmolemma of the jejunum absorbing enterocytes of cattle fetus in early fetal period

The data on the concentration of structural proteins of plasmolemma of the absorption enterocytes of the cattle fetus in the early fetal period are presented. Changes in the protein composition of the apical and basolateral membranes of enterocytes are manifested, characterized not only by changes in their ratio within different cell domains, but also by the dynamic redistribution of the number of different fractions of polypeptides between the poles of these cells. Analysis of the results of electrophoresis of apical membranes of the cattle fetus enterocytes in the early fetal period indicates a decrease in the content of low molecular weight protein fractions and an increase in high molecular weight. In the apical membranes of enterocytes of two-month-old cattle fetus, 25 protein fractions with a molecular weight of 9.6 to 205 kDa were detected. In the basolateral membranes revealed 23 protein fractions with a molecular weight of 9.6 to 120 kDa. High molecular weight fractions of polypeptides with molecular weights of 250 kDa and 300 kDa appear from the age of three months of embryos of calves. In the basolateral membranes of enterocytes, there is a slight decrease in the content of low molecular weight protein fractions, and from the four months of age of cattle fetus, high molecular weight fractions of polypeptides with molecular weights of 22.5 kDa and 155 kDa appear, which are absent in basolateral membranes of enterocytes. Two-month-old fetus lack proteins with a molecular weight of 19 kDa, 24 kDa, and 66 kDa in the apical part, whereas proteins with a mass of 22.5 kDa, 155 kDa, 170–185 kDa, and 205 kDa are absent on the basolateral membrane. In the apical membrane of enterocytes significantly more proteins with a low molecular weight of 9.6-14.2 kDa (1.56 times; P ≤ 0.001), whereas in the basolateral membrane a significantly higher concentration of proteins with a molecular weight of 15.5 kDa (2.06 times; P ≤ 0.001) and 17 kDa (3.62 times; P ≤ 0.001).

Structural proteins of plasmolemma of the jejunum absorbing enterocytes of cattle fetus in early fetal period

The data on the concentration of structural proteins of plasmolemma of the absorption enterocytes of the cattle fetus in the early fetal period are presented. Changes in the protein composition of the apical and basolateral membranes of enterocytes are manifested, characterized not only by changes in their ratio within different cell domains, but also by the dynamic redistribution of the number of different fractions of polypeptides between the poles of these cells. Analysis of the results of electrophoresis of apical membranes of the cattle fetus enterocytes in the early fetal period indicates a decrease in the content of low molecular weight protein fractions and an increase in high molecular weight. In the apical membranes of enterocytes of two-month-old cattle fetus, 25 protein fractions with a molecular weight of 9.6 to 205 kDa were detected. In the basolateral membranes revealed 23 protein fractions with a molecular weight of 9.6 to 120 kDa. High molecular weight fractions of polypeptides with molecular weights of 250 kDa and 300 kDa appear from the age of three months of embryos of calves. In the basolateral membranes of enterocytes, there is a slight decrease in the content of low molecular weight protein fractions, and from the four months of age of cattle fetus, high molecular weight fractions of polypeptides with molecular weights of 22.5 kDa and 155 kDa appear, which are absent in basolateral membranes of enterocytes. Two-month-old fetus lack proteins with a molecular weight of 19 kDa, 24 kDa, and 66 kDa in the apical part, whereas proteins with a mass of 22.5 kDa, 155 kDa, 170–185 kDa, and 205 kDa are absent on the basolateral membrane. In the apical membrane of enterocytes significantly more proteins with a low molecular weight of 9.6-14.2 kDa (1.56 times; P ≤ 0.001), whereas in the basolateral membrane a significantly higher concentration of proteins with a molecular weight of 15.5 kDa (2.06 times; P ≤ 0.001) and 17 kDa (3.62 times; P ≤ 0.001).

Fructose reabsorption by rat proximal tubules: role of Na+-linked cotransporters and the effect of dietary fructose

Fructose consumption has increased because of widespread use of high-fructose corn syrup by the food industry. Renal proximal tubules are thought to reabsorb fructose. However, fructose reabsorption (Jfructose) by proximal tubules has not yet been directly demonstrated, nor the effects of dietary fructose on Jfructose. This segment expresses Na+- and glucose-linked transporters (SGLTs) 1, 2, 4, and 5 and glucose transporters (GLUTs) 2 and 5. SGLT4 and -5 transport fructose, but SGLT1 and -2 do not. Knocking out SGLT5 increases urinary fructose excretion. We hypothesize that Jfructose in the S2 portion of the proximal tubule is mediated by luminal entry via SGLT4/5 and basolateral exit by GLUT2 and that it is enhanced by a fructose-enriched diet. We measured Jfructose by proximal straight tubules from rats consuming either tap water (Controls) or 20% fructose (FRU). Basal Jfructose in Controls was 14.1 ± 1.5 pmol·mm−1·min−1. SGLT inhibition with phlorizin reduced Jfructose to 4.9 ± 1.4 pmol·mm−1·min−1 ( P < 0.008), whereas removal of Na+ diminished Jfructose by 86 ± 5% ( P < 0.0001). A fructose-enriched diet increased Jfructose from 12.8 ± 2.5 to 19.3 ± 0.5 pmol·mm−1·min−1, a 51% increase ( P < 0.03). Using immunofluorescence, we detected luminal SGLT4 and SGLT5 and basolateral GLUT2; GLUT5 was undetectable. The expression of apical transporters SGLT4 and SGLT5 was higher in FRU than in Controls [137 ± 10% ( P < 0.01) and 38 ± 14% ( P < 0.04), respectively]. GLUT2 was also elevated by 88 ± 27% ( P < 0.02) in FRU. We conclude that Jfructose by proximal tubules occurs primarily via Na+-linked cotransport processes, and a fructose-enriched diet enhances reabsorption. Transport across luminal and basolateral membranes is likely mediated by SGLT4/5 and GLUT2, respectively.

Endothelial Tight Junctions Are Opened in Cholinergic-Evoked Salivation In Vivo

Blood vessels provide the original supplies for the formation of primary saliva, which is regulated by the tight junctions (TJs) between endothelial cells. Previous studies have shown that blood flow increases with vasodilatation during cholinergic-evoked salivation. However, changes in vascular paracellular permeability and the role of endothelial TJs in salivation are unknown. Here, we established an in vivo paracellular permeability detection system and observed that the endothelial TJs were permeable to 4-kDa fluorescein isothiocyanate (FITC)–dextran while impermeable to 40- and 70-kDa FITC-dextran under an unstimulated condition in mouse submandibular glands (SMGs). Pilocarpine increased the flux of 4- and 40-kDa FITC-dextran out of blood vessels but did not affect 70-kDa FITC-dextran. Claudin 5, a TJ protein specifically localized in salivary endothelial cells, was redistributed from the apicolateral membranes to the lateral and basolateral membranes and cytoplasm in cholinergic-stimulated mouse SMGs and freshly cultured human SMG tissues. In the transplanted SMGs from epiphora patients, we found that claudin 5 was present in the basolateral membranes and cytoplasm, instead of the apical region in control SMGs. Moreover, the level of phospho–myosin light chain 2 increased within the blood vessels of the pilocarpine-stimulated mouse SMGs and transplanted human SMGs, while the downstream molecule F-actin was reorganized in the endothelial cells of the transplanted human SMGs. Taken together, our findings provide direct visual evidence that the opening of endothelial TJs and the redistribution of claudin 5 are essential events contributing to cholinergic-evoked salivation, thus enriching our understanding of the secretory mechanisms that link blood flow to primary saliva formation by regulating the endothelial paracellular permeability.

Abstract 113: Renal Phenotype of Inwardly Rectifying Potassium Channel Kcnj16 (Kir 5.1) Knockout in the Dahl Salt-Sensitive Rats

The inward-rectifying channels play an important role in the control of resting membrane potential and tubular homeostasis in the kidney. Kcnj16 (Kir 5.1) form a heteromeric channel with Kcnj10 (Kir 4.1) at the basolateral membranes of aldosterone-sensitive distal nephron (ASDN); mutations in the human KCNJ10 gene result in SeSAME)/EAST syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. To illuminate the importance of Kcnj16 (Kir 5.1) in the context of a disease state in vivo, we generated a Kcnj16 knockout rat model in Dahl salt-sensitive (SS) background by using ZFN technology. ZFN against Kcnj16 caused a 18-bp in-frame deletion that occurred in the second protein transmembrane domain. IHC analysis demonstrated highly specific expression of Kcnj16 on the basolateral membranes of ASDN in the control kidneys of SS rats, which was completely abolished in Kcnj16-/- rats. The electrophysiological recording of K+ channels in the CCD basolateral membrane revealed activity of only homomeric Kcnj10 channels (21 pS channel in Kcnj16-/- rats compared to both 41 and 21 pS channels in SS rats). Thus, these data provide evidence of successful knock out of this protein and consequent degradation of the channel in renal tubules. The Kcnj16-/- knockout in SS rat induces electrolyte imbalance, epileptic seizures and result in changes in development (37% reduction in body and 54% in kidney mass). The mean arterial pressure was significantly lower in Kcnj16-/- compared to SS rats (91.3±1.8 to 104.7±5.5 mmHg) when animals were fed a low salt (0.4%) diet. Knockout of Kcnj16 resulted in hypokalemia (4.25±0.09 vs 2.08±0.12 mmol/L in serum of control vs KO rats), hypermagnesemia (0.49±0.02 vs 0.63±0.01 mmol/L in serum of control vs KO rats), and FSGS. Urea electrolyte balance was also disturbed compared to control animals. Importantly, change of the diet to high salt (4%) caused mortality of KO rats within 1-2 days. These data demonstrate critical role of Kcnj16 channels in renal salt handling and in the development of salt-sensitive hypertension.