Membrane Potential Of Epithelial Cells Research Articles

A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

Abstract 4024: Understanding the role of mitochondria in the progression from normal fibroblasts to DCIS associated fibroblasts

Abstract Background and Purpose: Ductal carcinoma in situ (DCIS) is a non-invasive breast cancer which may progress to invasive breast cancer. Many studies have focused on the epithelial cell mechanisms of progression from normal breast tissue to DCIS. However, in this study we want to study mechanisms of progression in cancer associated fibroblasts from normal breast tissue to DCIS. Loss of caveolin 1 (CAV1) in fibroblasts is a marker of reduced mitochondrial membrane potential and is a prognostic biomarker in ductal carcinoma in situ. High mitochondrial membrane potential in epithelial cells is a crucial determinant of carcinogenesis. Methods: In the study we use MCF10DCIS as a model for DCIS. MCF10DCIS.com (MCF10DCIS) is a clonal breast cancer cell line. The MCF10A, non-tumorigenic epithelial cell line, and human immortalized but non-transformed dermal fibroblasts (BJ1) were used study the difference between non-tumorigenic epithelial cells and DCIS and interactions between these two epithelial cells and fibroblasts. In order to study the interaction between epithelial cells and fibroblasts, we set up a coculture model system comprised both epithelial cells and fibroblasts. Immunofluorescence staining and confocal microscopy were used here to visualize the specific protein expression in different cell types. Results: (1) MCF10DCIS cells in coculture with fibroblasts have increased mitotracker red staining while as fibroblasts have decreased mitotracker staining compared to homotypic culture. However, coculture of MCF10A cells with fibroblasts did not change the mitotracker staining in either MCF10A or fibroblasts. (2) Loss of stromal CAV1 protein expression is observed in fibroblasts when fibroblasts are cocultured with MCF10DCIS cells compared to homotypic culture. CAV1 protein expression is unchanged in coculture with MCF10A cells. Conclusion: Previous studies have demonstrated that loss of stromal CAV1 and changes in mitochondrial membrane potential are factors involved in the transition from DCIS to invasive breast cancer. In this study we show that loss of stromal CAV1 and increases in epithelial mitochondrial membrane potential may play a role in the progression to DCIS. Citation Format: Ying-Hui Ko, Zhao Lin, Ubaldo E. Martinez Outschoorn. Understanding the role of mitochondria in the progression from normal fibroblasts to DCIS associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2015-4024

Plasma membrane depolarization‐induced ERK activation: role in regulating paracellular permeability of the proximal tubular epithelium

Both physiological and pathological conditions, including apical ion transport, hypoxia, oxidative stress, and ATP depletion can alter the membrane potential of epithelial cells. We have previously shown that membrane depolarization induces Rho‐Rho kinase (ROK) mediated phosphorylation of myosin light chain (MLC) in proximal tubule epithelial cells and elevates paracellular permeability. Here we show that the permeability increase was absent in cells stably expressing a non‐phosphorylatable, dominant negative MLC. Depolarization, elicited by high extracellular [K+],– or ouabain, an inhibitor of the Na+/K+ ATPase, also activated the Ras‐Raf‐MEK‐ERK pathway. ERK activation was fast, reversible and independent of intracellular Ca2+. Immunofluorescent staining revealed that ERK translocates to the cell periphery. The MEK inhibitor PD98059 reduced the depolarization‐induced rise in paracellular permeability. Interestingly, PD98059 also mitigated the depolarization‐induced MLC phosphorylation, raising the possibility that ERK affects paracellular permeability by contributing to increased contractility.In summary, our data suggest that cell contractility, regulated by the Rho‐ROK and the Ras‐ERK pathway might play a key role in epithelial barrier dysfunction caused by sustained depolarization.Support: NSERC, Banting Foundation and Kidney Foundation of Canada.

Nitric oxide-induced Cl- secretion in isolated rat colon is mediated by the release of thromboxane A2.

We have shown previously that thromboxane A2 (TXA2), which may be released by the anti-tumour drug irinotecan and by platelet-activating factor (PAF), causes Cl- secretion in the isolated rat colon. In the present study, the involvement of TXA2 in nitric oxide-induced Cl- secretion in isolated rat colon was investigated. In colonic mucosa set between Ussing chambers, the NO donor sodium nitroprusside (SNP; 100 microM) caused Cl- secretion, an effect that was almost completely inhibited by the NO scavenger carboxy-PTIO at 200 microM. The SNP-induced Cl- secretion was inhibited in a concentration-dependent manner by the TXA2 receptor antagonist ONO-3708 (IC50 = 2 microM) and the TX synthase inhibitor Y-20811 (IC50 = 0.4 microM). SNP significantly increased the release of TXA2 (measured as TXB2 release) from the mucosa. The SNP-induced increases in Cl- secretion and TXA2 release were blocked by a NO-sensitive guanylate cyclase inhibitor (ODQ). Dibutyryl cGMP (500 microM) also induced Cl- secretion, which was sensitive to ONO-3708 (10 microM) and Y-20811 (1 microM), and increased the release of TXA2 from the mucosa. PAF-induced (10 microM) Cl- secretion was inhibited by carboxy-PTIO (200 microM) and ODQ (10 microM), whereas irinotecan-induced (500 microM) Cl- secretion was not significantly inhibited by these drugs. A stable TXA2 analogue (STA2) but not SNP (100 microM) changed the membrane potential of epithelial cells in isolated colonic crypts under the whole-cell current-clamp condition. These results indicate that PAF elicits the NO-cGMP pathway and then stimulates the release of TXA2, which is a stimulant of colonic Cl- secretion. In contrast, the NO-cGMP pathway is not involved in the TXA2-mediated Cl- secretion induced by irinotecan.

Membrane potentials of epithelial cells in the epididymis of rats. Ionic and castration effects

1. 1. Membrane potentials were measured from rat epididymal epithelial cells in vitro at 35°C using micro-electrodes. The values obtained from the caput epididymis and the cauda epididymis were respectively −21.10 ± 0.26 mV (mean ± S.E. N = 470) and −30.53 ± 0.74 mV (mean ± S.E. N = 315). 2. 2. An increase in external potassium concentration depolarized the cells in both regions, a ten-fold increase in concentration causing a depolarization of 12mV in the caput and 22 mV in the cauda. 3. 3. Removal of sodium from the bathing solution caused a significant hyperpolarization in both regions as did treatment of the cells with amiloride (10 −4M). 4. 4. Ouabain (10 −3M) and 2,4-dinitrophenol (2.5 × 10 −4M) depolarized the cells in both regions. 5. 5. Castration caused a decrease in the membrane potentials which could be reversed by replacing rats with testosterone propionate (2 mg/kg per day), indicating that membrane potentials in the cauda epididymis were dependent on the supply of circulating androgens.

Effects of potassium ions and sodium ions on membrane potential of epithelial cells in rat duodenum

1. 1. Mucosal and serosal membrane potentials ( V m and V s) of epithelial cells in rat duodenum were recorded together with the transmural potential differences ( PD t). 2. 2. The value of V m in rat duodenum at 37°C was about −53 mV, being cosiderably greater than the values reported hitherto for the small intestine of various species. 3. 3. When Cl - in the mucosal medium was partially replaced with So 4 2- at fixed mucosal Na + and K + concentrations ([Na +] m and [K +] m), the membrane potential was affected in the steady state several minutes after replcement, whereas marked changes in the potential were observed with varying [K +] m or [Na +] m. 4. 4. As the mucosal K + concentration increased at constant [Na +] m, V m was gradually decreased (deploration), together with the increase in PD t. such a change in V m caused by varying [K +] m obeys Nernst's equation in the range of [K +] m higher than about 60 mm. 5. 5. At constant [K +] m, an increase in [Na +] m also caused the decrese of V m for the lower [k +] m region, whereas V m was not affected by such changes in [Na +] m in the range of [K +] m higher than approx. 60 mm. 6. 6. The values of P Na/ P k were obtained from the modified Goldman equation under an appropiate assumption. The ratio of the permeability coefficients markedly increases from zero to approx. 0.07 with a decrease in [K +] m.

Membrane potentials of epithelial cells in rat small intestine.

1. Stripped sacs of rat jejunum in which the outer muscle layers had been removed were found to maintain substantial transport and electrical activities.2. Mucosal and serosal membrane potentials of epithelial cells of normal and stripped everted sacs of rat jejunum were recorded in vitro together with the transmural potential difference.3. The cell interior was negative relative to both serosal and mucosal fluids, the transmural potential being the sum of the two membrane potentials.4. Changes in the transmural potentials in the presence of actively transferred hexoses and amino acids were entirely due to variations in the serosal potential, the mucosal potential being unchanged.5. Serosal and transmural potential increases on the addition of galactose were consistent with Michaelis-Menten kinetics, giving apparent K(m) values of 14.9 and 14.1 mM respectively.6. Phlorrhizin, ouabain, 2,4-dinitrophenol and sodium fluoroacetate inhibited serosal potential changes in the presence of galactose.7. Osmotic potentials resulting from transmural osmotic gradients originated from the serosal layers of the tissue.8. The results are consistent with the concept of a serosally located, electrogenic sodium pump which is stimulated by actively transferred hexoses and amino acids. The sodium-dependent entry mechanism at the mucosal membrane is non-electrogenic.

Ionic basis of membrane potentials of epithelial cells in rat small intestine.

1. Potentials across the mucosal and serosal membranes of the epithelial cells of rat jejunum together with transmural potentials were recorded using everted sac preparations.2. Ionic changes in either mucosal or serosal fluids affect mucosal or serosal membrane potentials respectively with comparable changes in the transmural potential. The contralateral membrane potential is relatively unaffected.3. Replacement of mucosal sodium chloride by potassium chloride or lithium chloride had little effect on potentials, but its replacement by mannitol or Tris chloride increased the negativity of the mucosal potential, giving linear relationships against log(10)[Na](m) with slopes of 41.4 and 30.7 mV respectively for tenfold change in [Na](m).4. At constant [Na](m), potassium or lithium increased the mucosal potential by 25.7 and 19.8 mV respectively for tenfold concentration changes.5. Qualitatively similar changes occurred in the serosal potential when the ionic composition of the serosal fluid was varied.6. Mucosal potential changes in response to modifications of the ionic composition of the mucosal fluid were the same in the presence and absence of galactose.7. Sodium and potassium diffusion potentials largely determine both the mucosal and serosal membrane potentials. For the mucosal membrane, P(K):P(Na) is 1.26:1, and is probably higher for the serosal membrane. Chloride makes no significant contribution to membrane potentials.8. Potentials generated by the electrogenic sodium pump are superimposed on diffusion potentials across the serosal membrane.

Experimental herpes simplex keratitis. Early alterations of corneal epithelium and stroma.

Epithelial and stromal changes in punctate lesions of rabbit corneas infected with herpes simplex virus (HSV) were studied by light and electron microscopy and measurements of epithelial intracellular and stromal potential differences were correlated with the histological observations. Virusinfected cells were enlarged, and the nuclear chromatin was marginated. Intracellular membrane potentials of epithelial cells in the punctate lesions were lower than normal. Nonenveloped viral particles were present in the nuclei of infected cells. Enveloped HSV particles were observed in inpocketings of the inner nuclear membrane, cytoplasmic tubules, extracellular spaces, and lying directly on basement membrane. The basement membrane was intact and of uniform thickness, even when polymorphonuclear leukocytes invaded the epithelial lesion. Numerous keratocytes with extensively developed endoplasmic reticulum were present in the superficial stroma immediately below the punctate lesions.