Nancy K. Wills

Basolateral membrane potential of a tight epithelium: ionic diffusion and electrogenic pumps.

SummaryThe contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K+)=72mm and (Cl−)=15.8mm. Cl− but not K+ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (PNa/PK) was 0.044, and the chloride to potassium permeability ratio (PCl/PK) was 1.17. Since K+ was not in electrochemical equilibrium, intracellular (K+) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K+ and Cl− the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na+. An equivalent electrical circuit for Na+ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.

Na+ channel activity in cultured renal (A6) epithelium: Regulation by solution osmolarity

SummarySolution osmolarity is known to affect Na+ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na+ currents (Isc).Isc for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringers solutions averaged 25±2, 9±2, 3±0.4, and 0.6±0.5 μA/cm2, respectively. Similar results were obtained following changes in the serosal tonicity; mucosal changes did not significantly affectIsc. The changes inIsc were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na+ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na+ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na+ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na+ transport rates for A6 epithelia reported in the literature.

Copper and zinc distribution in the human retina: Relationship to cadmium accumulation, age, and gender

The essential metals copper and zinc play vital roles in retinal cell survival and are crucial for the normal functioning of antioxidant enzymes. Retinal zinc deficiencies and decreased cellular antioxidative capacity have been linked to human retinal diseases including age-related macular degeneration (AMD). We recently reported that cadmium (a toxic metal with no known physiological function that interferes with copper and zinc metabolism) accumulates in human retinal tissues during aging. Moreover, cadmium content was higher in specific retinal tissues of aged women compared to men. Since cadmium, zinc and copper bind to similar proteins, we hypothesized that Cu and Zn content of human retinal tissues change as functions of cadmium accumulation during aging. Thus, we assessed the distribution of zinc and copper in the neural retina, retinal pigment epithelium (RPE) and choroid (Bruchs membrane-choroid; BMC) in male and female donors aged 1.5-87 years. Two independent methods, graphite furnace atomic absorption spectrometry and inductively-coupled plasma mass spectrometry, were used to measure Cd, Zn, and Cu in retinal tissues in human eyes from donors aged 1.5 to 87 years and the resulting values were normalized to protein concentration. Zn levels were approximately 5 times higher than Cu levels in the same tissues. The relative tissue distributions of these metals were: BMC>RPE>neural retina (Zn) and BMC>RPE=neural retina (Cu). In the choroid, mean Cu and Zn levels were higher in aged donors (>or=55 years old) than young donors (<55 years) and levels of these metals were strongly correlated with each other (r=0.90). In the neural retina, Cu and Zn both significantly decreased as a function of age. Several sex-related differences were found in the RPE. Specifically, copper levels were significantly higher in males than in females. In addition, both Zn and Cu levels in males were positively correlated with cadmium content, whereas this association did not occur in females. The results are consistent with co-regulation of zinc and copper stores in retinal tissues and suggest that the balance of these metals is associated with cadmium accumulation and gender. Thus, the roles of cadmium and gender differences in retinal metal balance warrant further investigation as factors in age-related retinal disease.

Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway

Cadmium (Cd), released from cigarette smoke and metal industrial activities, is known to accumulate in human body organs including retina and is particularly higher in retinal tissues of age-related macular degeneration (AMD) eyes compared to non-AMD eyes. We have determined the cytotoxic effects of Cd on human retinal pigment epithelial (RPE) cells. Upon Cd treatment, there was a dose- and time-dependent decline in ARPE-19 cell viability as well as early apoptotic changes such as altered mitochondrial membrane potential (MMP) and Cytochrome C release in cytosol. Depletion of GSH by buthionine-[S,R]-sulfoximine (BSO) resulted in increased Cd toxicity in ARPE-19 cells. Cadmium also caused reactive oxygen species (ROS) generation and activation of mitogen-activated protein kinases (MAPKs) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) significantly reduced Cd-induced toxicity. These results indicate that elevated ROS-induced activation of the MAPK signaling pathway could be associated with Cd-induced RPE cell apoptosis, one of the major contributing factors in AMD. The toxic effects of Cd on ARPE-19 cells indicate that environmental heavy metals such as Cd could be important potential factors in RPE cells death associated retinal diseases particularly related to smoking.

Cadmium-induced induction of cell death in human lens epithelial cells: implications to smoking associated cataractogenesis.

Cadmium is reported to accumulate in human eye tissues suggesting its implication in diverse ocular pathology. Using an in vitro cell culture model we investigated the effects of cadmium on human lens epithelial cells (HLECs) (HLE-B3). We observed cadmium-induced dose- as well as time-dependent decline in HLECs viability which was exacerbated significantly upon reduction of intracellular glutathione levels by buthionine sulfoximine (BSO). There was a dose-dependent significant increase in lactate dehydrogenase (LDH) release from HLECs suggesting cadmium-induced alteration of membrane integrity as well as necrotic cell death. The decline in cell viability was also due to apoptosis of the HLECs as determined by quantifying % apoptotic cells as well as PARP cleavage. Moreover, release of apoptosis inducing factor (AIF) into the cytosol was also detected. Cadmium was also observed to increase oxidative stress, lipid peroxidation and activation of MAPK pathway in HLECs. Antioxidants like N-acetylcysteine (NAC) and alpha-Tocopherol significantly prevented cadmium-induced toxicity in HLECs. Our findings suggest that cadmium-induced elevated oxidative stress as well as activation of MAPK signaling cascade eventually led to cell death of HLECs through apoptosis as well as necrosis. The loss of HLECs by cadmium could possibly explain its implication in cataract development particularly associated with smoking.

Current-voltage relationship of the basolateral membrane of a tight epithelium

The polyene antibiotic nystatin is used to reduce selectively to zero the apical membrane resistance of the rabbit descending colon, allowing the measurement of the current-voltage curve of the basolateral membrane. The I--V relationship is described by the Goldman-Hodgkin-Katz equations allowing calculation of PNa/PK, PCl/PK and PK for the basolateral membrane. Cs+ is found to block inward current (serosa to mucosa) in a manner similar to that found in excitable membranes.

Human retinal cadmium accumulation as a factor in the etiology of age-related macular degeneration

Cadmium is a naturally occurring, highly toxic, metallic element. It pollutes the environment as a result of industrial activity and accumulates in human tissues with a long biological half-life. Cadmium content has been demonstrated to increase in human retinal tissues as a function of age and tobacco smokers have approximately twice as much cadmium in retinal tissues than non-smokers. Smoking is also a key environmental risk factor for the retinal disease age-related macular degeneration (AMD). Recent studies have shown that urinary cadmium levels (a measure of Cd body burden) are higher in smokers who have AMD. We now report the Cd measurements in human retinal tissues from eyes afflicted with AMD compared to non-diseased eyes (controls) from age-matched donors. Human donor eyes frozen under argon gas were assessed for AMD severity using color stereoscopic fundus photographs and the Minnesota Grading System. Cadmium, zinc and, copper levels were measured in retinal tissues (neural retina, retinal pigment epithelium and choroid) using inductively coupled plasma mass spectrometry and graphite furnace spectrophotometry and values were normalized to tissue protein levels. Higher Cd levels were found in the neural retina and RPE for eyes afflicted with AMD compared to controls in males, differences were not statistically significant in females. The results indicate that higher retinal cadmium burdens are associated with the presence of AMD at least in males and suggest possible gender differences in the metabolism of metals in the human retina.

Osmotic regulation of Na+ transport across A6 epithelium: interactions with prostaglandin E2 and cyclic AMP.

Abstract. Previous work from this laboratory has shown that apical membrane sodium channel activity is stimulated by serosal hyposmotic solutions (Wills, Millinoff & Crowe, 1991). In the present study, we determined whether this stimulation of sodium transport is additive with the actions of prostaglandin E2 (PGE2) or cyclic AMP (cAMP).Addition of exogenous PGE2 (100 nm; serosal bath) to isosmotic solutions led to large increases in the amiloride-sensitive short-circuit current (Isc) and transepithelial conductance (Gt), whereas no significant effects of PGE2 were observed in hyposmotic serosal solutions. Subsequent addition of mucosal amiloride reduced Isc by ∼95% and Gt by ∼60%. Inhibition of endogenous PGE2 production by blockers of phospholipase A2 activity (quinacrine or 3[4-octadecyl]-benzoylacrylic acid; OBBA), or inhibition of cyclooxygenase activity by indomethacin reduced the stimulation of Isc and Gt by hyposmotic solutions. Addition of forskolin (FSK) or 3-Isobutyl-1-methylxanthine (IBMX) also resulted in approximately twofold increases in the amiloride-sensitive Isc and Gt and abolished the effects of subsequent hyposmotic challenge. The effects of forskolin, PGE2, and hyposmotic challenge were diminished by pretreatment with H89, a protein kinase A (PKA) inhibitor. We conclude that osmotic regulation of sodium channel activity interacts with multiple intracellular signaling pathways, specifically the arachidonic acid metabolic pathway and the cAMP/PKA intracellular messenger cascade.

Impedance analysis of epithelia

In view of the importance of epithelial organization in vectorial ion transport, it is clear that a complete understanding of regulation of this process requires an understanding of the role of the structural features involved. For example, although ion channels are the basis of much of the electrical activity and the electrochemical driving forces for ion transport across epithelial cell membranes, we can never truly understand this process without understanding the role of the lateral intercellular spaces or other structures such as microvilli and crypts in regulating extracellular ion concentrations. Similarly, in attempting to understand how the density of conducting channels is regulated, it is important to have knowledge concerning the role of membrane insertion or removal in regulating membrane transport properties.

A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope

In this study, we report two developments for studies of ion transport in cultured epithelial cells. First, a convenient method is presented for measuring apparent cell height using fluorescent microbeads as high-contrast landmarks of the apical and basal cell surfaces. The apparent cell height is then used as an indicator to monitor the time course of changes in cell volume in response to osmotic perturbations. Second, an Ussing-type chamber design for the inverted fluorescence microscope is presented, which allows determination of transepithelial electrical properties. Using these two methods, we obtained simultaneous measurements of cell height and transepithelial electrical parameters for cultured renal (A6) epithelium. Cell height was measured by alternately focusing the microscope between microbeads marking the apical and basal surfaces. The distance between these two surfaces was measured electrically from the voltage output of a potentiometer that was mechanically coupled to the fine-focusing knob of the microscope. Following decreases in the bathing solution osmolality, the cell height and transepithelial Na+ transport rate (measured as short-circuit current, ISC) increased. The increase in cell height preceded changes in ISC by several minutes, suggesting a lack of direct linkage between changes in cell volume and transepithelial Na+ transport. Both the fluorescent microbead cell height method and the Ussing-type chamber can be used in conjunction with patch-clamp techniques, intracellular microelectrode impalements, or fluorescent probes of intracellular composition. Therefore, this system may be advantageous for studies of epithelial cell volume and channel regulation.