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Item Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line(1996-08) Blazer-Yost, Bonnie; Record, Rae D.; Oberleithner, HansThe Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na+ transport.Item Gating-pore currents demonstrate selective and specific modulation of individual sodium channel voltage-sensors by biological toxins(ASPET, 2014-08) Xiao, Yucheng; Blumenthal, Kenneth; Cummins, Theodore R.; Pharmacology and Toxicology, School of MedicineVoltage-gated sodium channels are critical determinants of nerve and muscle excitability. Although numerous toxins and small molecules target sodium channels, identifying the mechanisms of action is challenging. Here we used gating-pore currents selectively generated in each of the voltage-sensors from the four α-subunit domains (DI-DIV) to monitor the activity of individual voltage-sensors and to investigate the molecular determinants of sodium channel pharmacology. The tarantula toxin huwentoxin-IV (HWTX-IV), which inhibits sodium channel current, exclusively enhanced inward gating-pore currents through the DII voltage-sensor. By contrast, the tarantula toxin ProTx-II, which also inhibits sodium channel currents, altered the gating-pore currents in multiple voltage-sensors in a complex manner. Thus, whereas HWTX-IV inhibits central-pore currents by selectively trapping the DII voltage-sensor in the resting configuration, ProTx-II seems to inhibit central-pore currents by differentially altering the configuration of multiple voltage-sensors. The sea anemone toxin anthopleurin B, which impairs open-channel inactivation, exclusively enhanced inward gating-pore currents through the DIV voltage-sensor. This indicates that trapping the DIV voltage-sensor in the resting configuration selectively impairs open-channel inactivation. Furthermore, these data indicate that although activation of all four voltage-sensors is not required for central-pore current generation, activation of the DII voltage-sensor is crucial. Overall, our data demonstrate that gating-pore currents can determine the mechanism of action for sodium channel gating modifiers with high precision. We propose this approach could be adapted to identify the molecular mechanisms of action for gating modifiers of various voltage-gated ion channels.Item Hormonal regulation of the epithelial Na+ channel: From amphibians to mammals(2006-05) Shane, Michael Anne; Nofziger, Charity; Blazer-Yost, BonnieHigh-resistance epithelia derived from amphibian sources such as frog skin, toad urinary bladder, and the A6 Xenopus laevis kidney cell line have been widely used to elucidate the underlying mechanisms involved in the regulation of vectorial ion transport. More recently, the isolation of high-resistance mammalian cell lines has provided model systems in which to study differences and similarities between the regulation of ion transporter function in amphibian and mammalian renal epithelia. In the present study, we have compared the natriferic (Na+ retaining) responses to aldosterone, insulin, and vasotocin/vasopressin in the A6 and mpkCCDcl4 (mouse principal cells of the kidney cortical collecting duct) cell lines. The functional responses of the epithelial Na+ channel (ENaC) to hormonal stimulation were remarkably similar in both the amphibian and mammalian lines. In addition, insulin- and aldosterone-stimulated, reabsorptive Na+ transport in both cell lines requires the presence of functional PI3-kinase.Item Modulation of basal and peptide hormone-stimulated Na transport by membrane cholesterol content in the A6 epithelial cell line(2005) West, Aaron; Blazer-Yost, BonnieThese studies examined the effect of altering plasma membrane cholesterol on basal Na+ flux as well as on the natriferic responses to the peptide hormones, insulin and anti-diuretic hormone (ADH) in the A6 model renal cell line. Membrane cholesterol concentrations were depleted or enriched using methyl-beta-cyclodextrin (MbetaCD) or a MbetaCD/cholesterol inclusion complex respectively. Effects of changes in the apical and basolateral plasma membranes were examined independently. Apical membrane cholesterol removal or supplementation had no effect on the basal Na+ transport rate. Short-term apical membrane cholesterol supplementation also had no effect on insulin-stimulated Na+ transport or on the initial phase of the ADH response. Interestingly, the additional apical membrane cholesterol had an inhibitory effect on the ADH response after 30 minutes. Apical membrane cholesterol depletion partially inhibited the responses to both insulin and ADH. Conversely, supplementation of basolateral cholesterol caused a significant increase in basal Na+ flux. Removal of cholesterol from the basolateral plasma membrane caused a decrease in basal Na+ flux with a time course analogous to channel turnover and completely inhibited peptide hormone responses. None of the changes in membrane cholesterol content decreased transcellular resistance. These results indicate an important role for membrane cholesterol content in the regulation of ENaC-mediated Na+ uptake.Item PPARγ agonists do not directly enhance basal or insulin-stimulated Na+ transport via the epithelial Na+ channel(2005-12) Nofziger, Charity; Chen, Lihong; Shane, Michael Anne; Smith, Chari D.; Brown, Kathleen K.; Blazer-Yost, BonnieSelective agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) are anti-diabetic drugs that enhance cellular responsiveness to insulin. However, in some patients, fluid retention, plasma volume expansion, and edema have been observed. It is well established that insulin regulates Na(+) reabsorption via the epithelial sodium channel (ENaC) located in the distal tubule. Therefore, we hypothesized that these agonists may positively modulate insulin-stimulated ENaC activity leading to increased Na(+) reabsorption and fluid retention. Using electrophysiological techniques, dose-response curves for insulin-mediated Na(+) transport in the A6, M-1, and mpkCCD(cl4) cell lines were performed. Each line demonstrated hormone efficacy within physiological concentration ranges and, therefore, can be used to monitor clinically relevant effects of pharmacological agents which may affect electrolyte transport. Immunodetection and quantitative PCR analyses showed that each cell line expresses viable and functional PPARgamma receptors. Despite this finding, two PPARgamma agonists, pioglitazone and GW7845 did not directly enhance basal or insulin-stimulated Na(+) flux via ENaC, as shown by electrophysiological methodologies. These studies provide important results, which eliminate insulin-mediated ENaC activation as a candidate mechanism underlying the fluid retention observed with PPARgamma agonist use.Item Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia(1998-04) Helman, Sandy I.; Liu, Xuehong; Baldwin, Kieron; Blazer-Yost, Bonnie; Els, Willem J.To study and define the early time-dependent response (< or = 6 h) of blocker-sensitive epithelial Na+ channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current (iNa) channel open probability (Po), and channel density (NT) under transient conditions of transport as measured by macroscopic short-circuit currents (Isc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 microA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. NT varied linearly over a 70-fold range of transport (0.5-35 microA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of Po occurred during control (10-20% over 2 h) and aldosterone experimental periods (10-30% over 6 h). When the Po of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, Po was observed to vary inversely with Isc, falling from approximately 0.5 to approximately 0.15 at the highest rates of Na+ transport or approximately 25% per 3-fold increase of transport. Because decreases of Po from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane NT.Item Upregulation of the sodium channel NaVβ4 subunit and its contributions to mechanical hypersensitivity and neuronal hyperexcitability in a rat model of radicular pain induced by local dorsal root ganglion inflammation(Ovid Technologies (Wolters Kluwer) - Lippincott Williams & Wilkins, 2016-04) Xie, Wenrui; Tan, Zhi-Yong; Barbosa, Cindy; Strong, Judith A.; Cummins, Theodore R.; Zhang, Jun-Ming; Pharmacology and Toxicology, School of MedicineHigh-frequency spontaneous firing in myelinated sensory neurons plays a key role in initiating pain behaviors in several different models, including the radicular pain model in which the rat lumbar dorsal root ganglia (DRG) are locally inflamed. The sodium channel isoform NaV1.6 contributes to pain behaviors and spontaneous activity in this model. Among all isoforms in adult DRG, NaV1.6 is the main carrier of tetrodotoxin-sensitive resurgent Na currents that allow high-frequency firing. Resurgent currents flow after a depolarization or action potential, as a blocking particle exits the pore. In most neurons, the regulatory β4 subunit is potentially the endogenous blocker. We used in vivo siRNA-mediated knockdown of NaVβ4 to examine its role in the DRG inflammation model. NaVβ4 but not control siRNA almost completely blocked mechanical hypersensitivity induced by DRG inflammation. Microelectrode recordings in isolated whole DRG showed that NaVβ4 siRNA blocked the inflammation-induced increase in spontaneous activity of Aβ neurons and reduced repetitive firing and other measures of excitability. NaVβ4 was preferentially expressed in larger diameter cells; DRG inflammation increased its expression, and this was reversed by NaVβ4 siRNA, based on immunohistochemistry and Western blotting. NaVβ4 siRNA also reduced immunohistochemical NaV1.6 expression. Patch-clamp recordings of tetrodotoxin-sensitive Na currents in acutely cultured medium diameter DRG neurons showed that DRG inflammation increased transient and especially resurgent current, effects blocked by NaVβ4 siRNA. NaVβ4 may represent a more specific target for pain conditions that depend on myelinated neurons expressing NaV1.6.