bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes
Authors: Hamm-Alvarez S. F. , Wei X. , Berndt N. , Runnegar M.
Published: December 2017

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.
Single-channel basis of slow inactivation of Na+ channels in rat skeletal muscle
Authors: Ruff R. L.
Published: December 2017

This study examined the single-channel basis of slow inactivation of Na+ currents (INa) in rat fast-twitch skeletal muscle fibers. A loose patch voltage clamp monitored changes in the maximum inward INa as the holding potential of the membrane patch changed. On a neighboring region of extrajunctional membrane of the same fiber, a gigaohm seal patch voltage clamp recorded single-channel INa. The maximum number of simultaneously open Na+ channels among a group of current traces indicated the maximum number of excitable channels. The holding potentials of the two voltage clamps were the same. Slow inactivation did not affect the open time or conductance of single Na+ channels. The number of excitable Na+ channels reversibly decreased during development of slow inactivation of INa and increased during recovery from slow inactivation of INa. Different stimulation protocols examined whether Na+ channels had to be in the closed, open, or fast-inactivated states to enter the slow-inactivated state. Na+ channels appear to be able to enter the slow-inactivated state from the closed, open, or fast-inactivated state.
Effects of cycloheximide on pancreatic endonuclease activity, apoptosis, and severity of acute pancreatitis
Authors: Kaiser A. M. , Saluja A. K. , Lu L. , Yamanaka K. , Yamaguchi Y. , Steer M. L.
Published: December 2017

The factors that determine the severity of acute pancreatitis are unknown, but a close inverse correlation between that severity and the extent of acinar cell apoptosis that follows the triggering signal has been previously noted [A. M. Kaiser, A. K. Saluja, A. Sengupta, M. Saluja, and M. L. Steer. Am. J. Physiol. 269 (Cell Physiol. 38): C1295-C1304, 1995]. In the present studies, we have evaluated internucleosomal DNA fragmentation and apoptosis within the pancreas and the effects of inhibiting protein synthesis by cycloheximide (CHX) on these phenomena as well as on the severity of pancreatitis. We report the constitutive presence of a Ca(2+)- and Mg(2+)-dependent endonuclease activity within pancreatic nuclei that is dependent on continued protein synthesis. Furthermore, we have found that CHX administration reduces the extent of apoptosis but significantly worsens the severity of pancreatitis that follows ligation of the rat common bile-pancreatic duct. These observations are consistent with the hypothesis that apoptosis is a teleologically beneficial response to acinar cell injury during acute pancreatitis.
Diacylglycerols derived from membrane phospholipids are metabolized by lipases in A10 smooth muscle cells
Authors: Migas I. , Severson D. L.
Published: December 2017

The metabolic fate of endogenous diacylglycerol (DAG) in cultured A10 smooth muscle cells was determined. Preincubation of A10 cells with [3H]myristic acid or [3H]arachidonic acid resulted in preferential labeling of phosphatidylcholine (PC) or phosphatidylinositol (PI), respectively. Addition of PC-specific phospholipase C (PC-PLC) to [3H]myristate-labeled A10 cells resulted in a 10-fold increase in radiolabeled DAG, which was converted to monoacylglycerol (MG) and fatty acid (FA). DAG degradation and MG formation was inhibited by tetrahydrolipstatin, a DAG lipase inhibitor. PC-derived DAG was not converted to phosphatidic acid; in addition, PC resynthesis or triacylglycerol synthesis was not observed. Addition of PI-specific PLC (PI-PLC) to [3H]arachidonate-labeled A10 cells resulted in a modest increase in radiolabeled DAG that was also hydrolyzed to MG and FA. Therefore, the principal metabolic fate of endogenous DAG generated from membrane phospholipids by treatment of A10 cells with PC-PLC and PI-PLC was hydrolysis by a DAG lipase pathway.
Intracellular pH regulation in detubulated frog skeletal muscle fibers
Authors: Putnam R. W.
Published: December 2017

Intracellular pH regulation was studied in semitendinosus muscle fibers from frog (Rana pipiens). Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes and membrane potential with conventional microelectrodes. Fibers had their connections between the surface and transverse tubular membrane disrupted (detubulation) with the formamide shock technique. Fibers were approximately 80% detubulated as determined by the decrease in membrane capacitance and the loss of contractile capability. The initial rate of pHi recovery from acidification to approximately 6.8 (no CO2) was dependent on external buffering power, reaching a maximum of approximately 0.6 pH/h at 50 mM HEPES, indicating that the rate of pHi recovery in frog muscle is limited by the diffusion of buffer through an external "unstirred layer". In detubulated fibers, pHi recovery from acidification due to both the amiloride-sensitive Na+/H+ and the 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive (Na+ + HCO3-)/Cl- exchangers was nearly identical to recovery in fully tubulated fibers. This is consistent with these two pH recovery transporters being localized to the surface, and not the transverse tubular, membrane domain in frog skeletal muscle fibers.
Mercuric chloride uncouples glutamate uptake from the countertransport of hydroxyl equivalents
Authors: Nagaraja T. N. , Brookes N.
Published: December 2017

The cotransport of sodium and glutamate by system X(AG)- is believed to be coupled to the countertransport of potassium and hydroxyl ion equivalents. Accordingly, the uptake of glutamate or D-aspartate in astrocytes is accompanied by an intracellular acidification. Here, we report that HgCl2 blocks the glutamate-induced acidification with an approximate 50% inhibitor concentration (IC50) of 55 nM, an order of magnitude below its IC50 for inhibition of glutamate uptake. At 100 nM HgCl2, glutamate-induced acidification was abolished, whereas glutamate uptake was unaffected. D-Aspartate-induced acidification was equally sensitive to HgCl2, indicating that HgCl2 blocked a transporter-mediated, rather than a receptor-mediated, acidification. Unaltered responses to acute acid and alkaline loads showed that HgCl2 was not acting indirectly via a change in pH regulation. We conclude that HgCl2 acted directly on the glutamate transporter to uncouple the uptake of glutamate from the export of hydroxyl equivalents. In contrast, two other sulfhydryl reagents, p-chloromercuribenzensulfonate and N-ethylmaleimide, failed to discriminate between glutamate-induced acidification and glutamate uptake. An additional effect of > or = 100 nM HgCl2, in this case shared by p-chlormercuribenzenesulfonate, was transient intracellular acidification. There is evidence that glutamate transport is regulated by intracellular pH. Mercuric mercury may disrupt the regulation of glutamate transport at lower concentrations than those that block transport.
Expression of CFTR controls cAMP-dependent activation of epithelial K+ currents
Authors: Loussouarn G. , Demolombe S. , Mohammad-Panah R. , Escande D. , Baro I.
Published: December 2017

The perforated-patch configuration of the patch-clamp technique was used to record whole cell currents from human epithelial CFPAC-1 cells defective for functional cystic fibrosis transmembrane conductance regulator (CFTR). In CFPAC-1 cells, adenosine 3',5'-cyclic monophosphate (cAMP) stimulation with forskolin (10 microM) plus 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) activated neither Cl- nor K+ currents. In the same cells transfected with wild-type CFTR gene, cAMP stimulation produced activation of both Cl- and K+ currents. In Cl(-)-depleted medium (gluconate as a substitute), cAMP stimulation evoked a K+ current in CFTR-transfected but not in untransfected CFPAC-1 cells. This cAMP-evoked K+ current was the sum of two components: 1) a time-independent inwardly rectifying component, and 2) a slowly relaxing component activated at positive voltages. Increasing intracellular Ca2+ with ionomycin (1 microM) activated K+ currents in either transfected or untransfected cells. In transfected cells, blocking the CFTR conductance with high-concentration glibenclamide (100 microM) reduced the K+ current when activated by cAMP but not when activated by Ca2+. Pretreating CFTR-transfected cells for 48 h with interferon-gamma downregulated CFTR gene expression and reduced cAMP but not Ca2+ activation of the whole cell K+ current. From these results, we conclude that functional membrane CFTR protein influences activation by cAMP of epithelial K+ currents.
Mechanism of activation by cGMP-dependent protein kinase of large Ca(2+)-activated K+ channels in mesangial cells
Authors: Stockand J. D. , Sansom S. C.
Published: December 2017

The patch clamp method was employed to establish the mechanism of regulation by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) of large Ca(2+)-activated K+ channels (BKCa) in human mesangial cells. Dibutyryl cGMP (DBcGMP) significantly increased open probability (Po) of BKCa in the absence but not in the presence of staurosporine in cell-attached patches. In excised patches, BKCa was activated by simultaneous addition of MgATP plus cGMP but not cAMP plus MgATP. Activation by cGMP plus MgATP was blocked by KT-5823, an inhibitor of PKG, but not by KT-5720, an inhibitor of cAMP-dependent protein kinase (PKA). Thus a cGMP-specific endogenous kinase is associated with mesangial BKCa. In excised patches, exogenous PKG but not PKA or protein kinase C activated BKCa. The half-activation potential (V1/2), defined as the potential at which the Po = 0.5 with 1 microM Ca2+, was -34 and 42 mV for activated and inactivated BKCa, respectively; however, the gating charge (Zg), a measure of voltage sensitivity, was not affected by PKG. Similarly, the Ca1/2 (free Ca2+ concentration required to activate to Po = 0.5 at 40 mV) decreased from 1.74 to 0.1 microM on addition of PKG, but the Hill coefficient, a measure of Ca2+ sensitivity, was not affected. Activation of BKCa by PKG was heterogeneous with two populations: the majority (67%) activated by PKG and the minority unaffected. It is concluded that an endogenous PKG activates BKCa by decreasing the Ca2+ and voltage activation thresholds independently of sensitivities.
Caerulein-stimulated arachidonic acid release in rat pancreatic acini: a diacylglycerol lipase affair
Authors: Hou W. , Arita Y. , Morisset J.
Published: December 2017

This study was performed to evaluate the effect of caerulein, a cholecystokinin analogue, on arachidonic acid (AA) release in rat pancreatic acini and to determine the cellular mechanism involved. Caerulein did not stimulate phospholipase A2 (PLA2); however, diacylglycerol (DAG) lipase activity was increased. Validity of PLA2 or DAG lipase inhibitors was confirmed by their ability to selectively inhibit PLA2 or DAG lipase activities. Caerulein increased AA release from acini prelabeled with [3H]AA both dose and time dependently. Inhibitors were used to evaluate the involvement of different signaling pathways. Mepacrine and aristolochic acid, two PLA2 inhibitors, did not inhibit caerulein-induced AA release, whereas the DAG lipase inhibitor RHC-80267 did. The phospholipase C (PLC) inhibitor U-73122 totally inhibited caerulein-induced AA release, whereas the phospholipase D (PLD) inhibitor wortmannin had no effect. Our data indicate that caerulein-induced AA release results from the combined action of PLC and DAG lipase without PLA2 or PLD activation.
Mechanisms of inhibition of insulin release
Authors: Sharp G. W.
Published: December 2017

Several agonists including norepinephrine, somatostatin, galanin, and prostaglandins inhibit insulin releasse. The inhibition is sensitive to pertussis toxin, indicating the involvement of heterotrimeric Gi and/or Go proteins. Receptors for the different agonists have different selectivity for these G proteins. After G protein activation, the alpha- and beta gamma-subunits dissociate and interact with multiple targets to inhibit release. These include 1) the ATP-sensitive K+ channel and perhaps other K+ channels, 2) L-type voltage-dependent Ca2+ channels, 3) adenylyl cyclase, and 4) a “distal” site late in stimulus-secretion coupling. The latter effect, which may be exerted close to the final stage of exocytosis, is the most powerful of the individual inhibitory mechanisms. G protein action on the target molecules is determined by the individual G proteins activated and their specificity for the targets. The L-type Ca2+ channel is inhibited by G(o)-1. Adenylyl cyclase is inhibited by Gi-2 and Gi-3. The distal inhibition can be exerted by Gi-1, Gi-2, Gi-3, and G(o)-2. Thus there is both selectivity and promiscuity in G protein action in the beta-cell. These characteristics allow an inhibitory ligand to be effective at multiple targets and to act differentially from other inhibitory ligands.
Effects of lost surface area on red blood cells and red blood cell survival in mice
Authors: Waugh R. E. , Sarelius I. H.
Published: December 2017

The effects of removing area from mouse red blood cells on the fate of the cells after reinfusion were investigated. When cells were made nearly spherical (by reducing cell area by approximately 35%) and then reinfused into the animal, most were cleared from the circulation within 1-2 h, although approximately 20% of the cells survived for 4 h or longer. When only 20% of the area was removed (leaving a 15% excess), more than 90% of the cells continued to circulate for 4 h. After reinfusion, the mean surface area of the surviving cells remained constant (73-75 microns2), but the mean volume decreased, from 56.6 +/- 2.1 to 19.1 +/- 1.5 microns3 (+/- SD of 5 replicates) over 4 h. These changes did not occur in cells suspended in plasma but not reinfused into the animal. Thus a loss of surface area results in a decrease in cell volume, as if to maintain a requisite degree of deformability. The results support the hypothesis that the increase in cell density associated with increasing cell age may be a consequence of surface area loss.
Rapid effects of steroid hormones on free intracellular calcium in T84 colonic epithelial cells
Authors: Doolan C. M. , Harvey B. J.
Published: December 2017

Studies from our laboratory have demonstrated rapid (< 1 min) nongenomic activation of K+ recycling and Na+/H+ exchange by mineralocorticoids in human colonic epithelium, and studies from other laboratories have demonstrated rapid effects of aldosterone on intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells. Here a rapid nongenomic effect of aldosterone on [Ca2+]i is demonstrated in the human colonic epithelial cell line T84. Aldosterone induced a rapid increase in [Ca2+]i within approximately 2 min. The rise in [Ca2+]i after aldosterone appears to result from the activation of a Ca2+ influx pathway, inasmuch as 1) no increase in [Ca2+]i was observed with aldosterone when cells were bathed in Ca(2+)-free Krebs solution and 2) emptying of the intracellular Ca2+ stores by thapsigargin was not enhanced by addition of aldosterone to extracellular Ca(2+)-free solution. In contrast, the Ca2+ response to aldosterone, in the presence of 2 mM Ca2+ in the external bathing solution, was not decreased after intracellular Ca2+ stores were emptied by thapsigargin. Other mineralocorticoid hormones increased [Ca2+]i, whereas the glucocorticoid hydrocortisone failed to increase [Ca2+]i. These results demonstrate the existence of a mineralocorticoid-specific Ca(2+)-signaling pathway in human colonic T84 (crypt) epithelial cells.
L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture
Authors: Nowak G. , Schnellmann R. G.
Published: December 2017

The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.
Noncoordinated expression of alpha-, beta-, and gamma-subunit mRNAs of epithelial Na+ channel along rat respiratory tract
Authors: Farman N. , Talbot C. R. , Boucher R. , Fay M. , Canessa C. , Rossier B. , Bonvalet J. P.
Published: December 2017

Na+ reabsorption from the epithelial surface of the respiratory tract plays a fundamental role in respiratory physiology. As in the epithelia of the renal collecting tubule and distal colon, Na+ enters across the luminal surface of respiratory epithelial cells via a recently cloned amiloride-sensitive multisubunit (alpha, beta, gamma) epithelial Na+ channel. We have examined the cellular expression at the mRNA level of the alpha-, beta-, and gamma-subunits of rat epithelial Na+ channel (rENaC) in the rat lung and upper airway epithelial cells using in situ hybridization. A large prevalence of alpha- and gamma-rENaC subunit expression (over beta) was found in tracheal epithelium, in a subpopulation of alveolar cells, presumably type II pneumocytes, and in nasal and tracheal gland acini. In contrast, equivalent levels of expression of all three subunits were detected in bronchiolar epithelium and in rat nasal gland ducts. This diversity of expression may reflect cell-specific functions of the amiloride-sensitive Na+ channel along the respiratory tract.
Extracellular Ca2+ directly regulates tight junctional permeability in the human cervical cell line CaSki
Authors: Gorodeski G. I. , Jin W. , Hopfer U.
Published: December 2017

Lowering extracellular calcium concentration ([Ca2+]o) increases acutely and reversibly the transepithelial electrical conductance (G(TE)) and the epithelial permeability to pyranine (Ppyr) across CaSki cultures. Effects were already observed after lowering calcium from 1.2 to 1.0 mM and were maximal at 0.1 mM. The dose-response curves were sigmoidal (calcium concentration that produces half-maximal effect = 0.3 mM), and the time courses indicated simple exponential trends (time constants of 4-5 min). The effect of calcium was not mediated by mobilization of cytosolic calcium or altering calcium influx, and manganese was found to be a partial agonist to [Ca2+]o. The effects of [Ca2+]o, on permeability were additive to those of hypertonic conditions, indicating that calcium modulates junctional permeability. The experimental data were fitted to theoretical models that relate changes in G(TE) to the probability of assembled/disassembled tight junctions. The results suggest that calcium interacts directly and cooperatively at extracellular sites with junctional elements that are arranged in parallel, and it shifts the probability state of the junctions from "open" to "closed" state. Changes in extracellular calcium may affect the permeability of tight junctions of the cervical epithelium and may play a role in regulating production of cervical mucus.
Roles of Ca2+ and Na+ in the inward current and action potentials of guinea pig ureteral myocytes
Authors: Sui J. L. , Kao C. Y.
Published: December 2017

Physiological roles of Ca2+ vs. Na+ in membrane currents and action potentials of ureteral myocytes were investigated on freshly dissociated guinea pig ureteral myocytes with the patch-clamp method. The myocytes are spindle shaped, with cell volume of 2,473 microm3, surface area of 2,014 microm2, capacitance of 48.2 pF, resting potential of -47.9 mV, and membrane conductance of 840 pS. The membrane current consists of a slow inward Ca2+ current (ICa) conducted by L-type Ca2+ channels and an actively fluctuating Ca2+-activated K+ current [IK(Ca)] conducted by Ca2+-activated maxi-K+ channels. ICa dominates the membrane current by being long lasting and more active at less depolarized potentials than IK(Ca) and by regulating IK(Ca). Ca2+-free media, Co2+, and nifedipine reduce or block ICa, whereas high extracellular Ca2+ concentration and BAY K 8644 enhance it. Action potential amplitudes and plateaus are regulated correspondingly. Related changes are also seen in IK(Ca) In contrast, no fast inward current attributable to Na+ was found. Replacing extracellular Na+ with tris(hydroxymethyl)aminomethane had no apparent effects on the inward or outward current or on the action potentials.
Properties of inward calcium current in guinea pig ureteral myocytes
Authors: Sui J. L. , Kao C. Y.
Published: December 2017

Ureteral myocytes of guinea pigs have L-type Ca2+ channels (I(Ca)). In 3 mM Ca2+, maximum I(Ca) was 3.38 microA/cm2. Voltage at which conductance is 50% of maximum (V0.5) of I(Ca) was -1.0 mV in 3 mM Ca2+ and +22 mV in 30 mM Ca2+, with slope factors of 8 mV. V0.5 of steady-state inactivation of I(Ca) was -16.2 and +1.1 mV in 3 and 30 mM Ca2+, respectively, with similar slope factors of about -6 mV. A window current reaching 20-25% of the maximum I(Ca) was active between -20 and 0 mV. I(Ca) inactivated very slowly, with time constants of 217.6 and 2,455.9 ms with no voltage dependency. When Ba2+ was used as the charge carrier, the amplitude and inactivation kinetics of the Ba2+ current were similar to those for I(Ca). These results indicate that the ureteral myocyte has little Ca2+-mediated Ca2+ channel inactivation, a feature significantly associated with the slow I(Ca) inactivation. The slow inactivation and the window current are essential for the sustained membrane depolarization during the plateau of ureteral action potentials.
cAMP stimulates CFTR-like Cl- channels and inhibits amiloride-sensitive Na+ channels in mouse CCD cells
Authors: Letz B. , Korbmacher C.
Published: December 2017

Confluent M-1 mouse cortical collecting duct (CCD) cells express highly selective low-conductance amiloride-sensitive Na+ channels (B. Letz, A. Ackermann, C. M. Canessa, B. C. Rossier, and C. Korbmacher, J. Membr. Biol. 148: 129-143, 1995). Here we investigated the effect of forskolin on membrane voltage and whole cell currents of confluent M-1 cells using the patch-clamp technique. Forskolin (1 microM) reduced the hyperpolarization in response to amiloride (10 microM) from 17 to 4 mV and decreased the amiloride-sensitive Na+ inward currents from 81 to 26 pA. Furthermore, forskolin increased the hyperpolarization caused by changing from an apical low-Cl- solution (9 mM) to a high-Cl- solution (149 mM) from 11 to 30 mV and increased the magnitude of the inward current changes induced by alternating between high-Cl- and low-Cl- solutions from 25 to 138 pA. This demonstrates that forskolin stimulates an apical Cl- conductance. Anion substitution experiments revealed a permeability sequence SCN- > Br- > Cl- > I- >> gluconate. This suggests that the stimulated channels are cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl- channels. 3-Isobutyl-1-methylxanthine and 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate mimicked the effects of forskolin, whereas 1,9-dideoxyforskolin had no effect. We conclude that, in addition to amiloride-sensitive Na+ channels, CFTR-like Cl- channels are present in the apical membrane of confluent M-1 cells. An increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) activates these Cl- channels and concurrently reduces the activity of the Na+ channels. This reciprocal regulation by cAMP suggests that the channels are functionally coupled.
Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion
Authors: Sangan P. , Rajendran V. M. , Mann A. S. , Kashgarian M. , Binder H. J.
Published: December 2017

Active K absorption in the rat distal colon is energized by an apical membrane H-K-ATPase, whereas K absorption in the distal collecting duct is generally believed to be modulated by a related renal H-K-ATPase. Experiments were performed to establish the mechanism(s) by which dietary Na depletion (with resulting elevated aldosterone levels) and K depletion stimulate K absorption. A colonic H-K-ATPase-specific cDNA probe and a polyclonal antibody were utilized to measure mRNA (Northern blot analyses) and protein (Western blot and immunofluorescence studies) abundance in the distal and proximal colon and renal collecting ducts and cortex of dietary Na- and K-depleted rats. Dietary Na depletion, but not K depletion, upregulated H-K-ATPase-specific mRNA and protein expression in the distal and proximal colon; Na depletion also stimulated H-K-ATPase activity in the distal colon. In contrast to the distal colon, H-K-ATPase-specific protein level in the outer medulla was enhanced by dietary K depletion, but not by Na depletion. This study establishes that 1) dietary Na depletion stimulates colonic H-K-ATPase activity most likely by a transcriptional process and 2) the regulation of colonic H-K-ATPase expression by dietary Na depletion and dietary K depletion is not identical in the large intestine and differs in the kidney from the colon, suggesting the presence of two (or more) H-K-ATPase isoforms in the rat colon.
Intracellular regulation of intestinal folate uptake: studies with cultured IEC-6 epithelial cells
Authors: Said H. M. , Ma T. Y. , Ortiz A. , Tapia A. , Valerio C. K.
Published: December 2017

Although the mechanism of folate uptake in the small intestine has been well characterized, very little is known about the intracellular regulation of the uptake process. Using mature confluent monolayers of the intestinal epithelial cell line IEC-6 as an in vitro intestinal epithelial cell model, we have found the uptake of folic acid to be similar to that of the native small intestine in that it is 1 ) temperature, energy, and pH dependent, 2) Na+ independent, 3) inhibited by structural analogs and anion transport inhibitors, and 4) saturable as a function of substrate concentration [apparent Michaelis constant (Km) = 0.45 +/- 0.06 microM; maximal velocity (Vmax) = 3.08 +/- 0.14 pmol x mg protein(-1) x 5 min(-1)]. Furthermore, IEC-6 cells were found by Northern blot analysis to lack the expression of the membrane folate-binding protein. Pretreatment of IEC-6 monolayers with specific protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin A25 caused a significant inhibition in folic acid uptake. On the other hand, their negative controls, genistin and tyrphostin A1, respectively, had no effect. The inhibitory effect of genistein was mediated through inhibition in the Vmax of the folate uptake process with no change in the apparent Km. Pretreatment of IEC-6 monolayers with compounds that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level (e.g., dibutyryl cAMP) also resulted in a significant (though modest) inhibition in folic acid uptake; however, specific inhibitors of protein kinase A did not affect the uptake process. Specific modulators of protein kinase C and Ca2+/calmodulin-mediated pathways did not significantly affect folic acid uptake. These results demonstrate the suitability of IEC-6 monolayers as an intestinal epithelial model to study folate transport and demonstrate for the first time that uptake of folic acid is regulated by a PTK- and a cAMP-mediated pathway.
Stem cell factor alters membrane potential of purified peritoneal mast cells in culture
Authors: Wang Y. H. , Go V. L. , Wu S. V. , Wei J. Y.
Published: December 2017

The membrane potential (E(m)) was used as an indicator to evaluate the effect of stem cell factor (SCF) on the membrane integrity of peritoneal mast cells (PMCs). PMCs were harvested from the peritoneal lavage of Sprague-Dawley rats, purified more than 95% and cultured with or without the presence of SCF (2 x 10(-8) M). E(m) values were measured with conventional intracellular recording techniques. Results from day 1 to day 4 in culture were compared. Significant differences in average E(m) (aE(m)) (P < 0.01, analysis of variance) were seen on days 3 and 4 (means +/- SE in millivolts): -67.4 +/- 8.0 and -59.4 +/- 4.8 with SCF vs. -24.8 +/- 7.9 and -7.6 +/- 3.9 without SCF, respectively. Moreover, after culture with SCF for > 1 wk, the aE(m) values of purified PMCs had a tendency to reach plateau values similar to that of unpurified PMCs on day 1 (at -20 mV). The morphological appearances of PMCs can be correlated with the results of aE(m) measurements. PMCs with a smooth spherical shape and highly refractive appearance, and better tolerance to electrode impalement, showed E(m) with greater negative values and lesser fluctuations. These results indicate that SCF can maintain the membrane properties and viability of purified PMCs in a long-term culture.
The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities
Authors: Blazer-Yost B. L. , Helman S. I.
Published: December 2017

The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+ transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.
Mechanism of vasopressin-induced increase in intracellular Ca2+ in LLC-PK1 porcine kidney cells
Authors: Dibas A. I. , Rezazadeh S. M. , Vassan R. , Mia A. J. , Yorio T.
Published: December 2017

Analysis of the signal transduction cascade of vasopressin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in LLC-PK1 cells was performed. First, a comparison of the effect of vasopressin on [Ca2+]i in LLC-PK1 cells with that produced in rat hepatocytes was performed [an intracellular mobilizing mechanism involving a V1 receptor coupled to the production of inositol 1,4,5-trisphosphate (IP3)]. Second, the effect of known inhibitors of intracellular Ca2+ mobilization on vasopressin Ca2+ response in LLC-PK1 cells was studied. Vasopressin induced a transient increase in [Ca2+]i in both LLC-PK1 cells and hepatocytes. In contrast to the single [Ca2+]i spike seen in LLC-PK1 cells, vasopressin induced an average of two to three [Ca2+]i spikes in hepatocytes. The V1 antagonist (Pmp1-O-Me-Tyr2-[Arg8]vasopressin, 1 microM) abolished vasopressin Ca2+ response in both cell types. Inhibitors of intracellular Ca2+ mobilization, thapsigargin (5 microM) and U-73122 (3 microM), abolished the Ca2+ response by vasopressin in LLC-PK1 cells. The results suggest that vasopressin-induced increase in [Ca2+]i in LLC-PK1 cells is mediated via a V1-like receptor and involves the mobilization of intracellular Ca2+ through an IP3- or thapsigargin-sensitive Ca2+ pool.
A possible role of inwardly rectifying K+ channels in chick myoblast differentiation
Authors: Shin K. S. , Park J. Y. , Kwon H. , Chung C. H. , Kang M. S.
Published: December 2017

We examined the developmental change of inwardly rectifying K+ channels (IRK) and its possible role in myogenesis. Northern blot analysis revealed an increase in the level of IRK mRNA during myogenesis. Accordingly, IRK current was not detectable in replicating myoblasts but first appeared in aligned myoblasts that were competent for fusion and gradually increased thereafter. The time course change of IRK activity was closely related to the increase in resting membrane potential during myogenesis. Application of 0.5 mM Ba2+ to the bath depolarized the membrane and blocked IRK currents dramatically but not outwardly rectifying K+ currents. Myoblasts devoid of IRK had low resting K+ permeability, whereas myotubes that possess IRK had high resting K+ permeability. In some aligned myoblasts, anomalous hyperpolarization was elicited by increasing extracellular K+ concentration, which may be attributable to the increased conductance of IRK. Noteworthy was the fact that maximal fusion was obtained at this range of K+ concentration. These findings imply that IRK is responsible for the change in the K+ permeability during chick myogenesis, which may provide a larger driving force for Ca2+ influx that is a prerequisite for myoblast fusion.
Cyclic nucleotide-gated cation channels mediate sodium absorption by IMCD (mIMCD-K2) cells
Authors: Vandorpe D. H. , Ciampolillo F. , Green R. B. , Stanton B. A.
Published: December 2017

The inner medullary collecting duct cell line, mIMCD-K2, absorbs Na+ by an amiloride-sensitive, electrogenic mechanism. The goal of the present study was to characterize the amiloride-sensitive, Na+ -conducting channels responsible for electrogenic Na+ absorption. To this end, we measured Na+ currents in single cells with the patch-clamp technique and Na+ currents across monolayers mounted in Ussing-type chambers. In whole cell patch-clamp experiments, amiloride-sensitive, inward Na+ currents were mediated by nonselective cation channels. In single-channel patch-clamp experiments, amiloride- and guanosine 3',5'-cyclic monophosphate (cGMP)-sensitive, 20-pS nonselective cation channels (i.e., CNG channels) were identified in the apical membrane. CNG channels were inhibited by amiloride, diltiazem, ethylisopropylamiloride (EIPA), and 8-bromo-cGMP and were permeable to Ca2+ and Mg2+. Epithelial Na+ channels were never observed in whole cell or single-channel recordings. Na+ absorption across confluent monolayers was inhibited with a rank order potency of benzamil > amiloride > phenamil >> EIPA > diltiazem. Our data are most consistent with the view that CNG channels mediate electrogenic Na+ absorption across mIMCD-K2 cells.
Na+-K+-ATPase alpha-subunit containing Q905-V930 of gastric H+-K+-ATPase alpha preferentially assembles with H+-K+-ATPase beta
Authors: Wang S. G. , Eakle K. A. , Levenson R. , Farley R. A.
Published: December 2017

Amino acids N886-A911 of the rat Na+-K+-ATPase alpha3-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H+-K+-ATPase alpha-subunit. The chimera (NGH26) was expressed in yeast with the rat Na+-K+ -ATPase beta1-subunit (rbeta1), the rat H+-K+-ATPase beta-subunit (HKbeta), the chimeric beta-subunit NHbeta1 (containing the carboxy-terminal ectodomain of HKbeta), or the chimeric beta-subunit HNbeta1 (containing the carboxy-terminal ectodomain of rbeta1). Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HKbeta and NHbeta1 rather than with rbeta1 or HNbeta1. Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HKbeta or NHbeta1. These results suggest that the sequence Q905-V930 interacts with the HKbeta-subunit on the extracellular side of the cell membrane. The NGH26 + HKbeta complex is less stable than alpha3 + HKbeta when heated and also has a lower binding affinity for ouabain [dissociation constant (Kd) = 63 nM] compared with alpha3 + rbeta1 or alpha3 + HKbeta (K(d) = 5-10 nM). In contrast, the NGH26+NHbeta1 complex is thermally as stable as alpha3 + rbeta1 complexes, and its ouabain binding affinity (K(d) = 10 nM) is the same as the wild type. These results indicate that the amino acids Q905-V930 of the rat gastric H+-K+-ATPase alpha-subunit preferentially associate with the extracellular domain of H+-K+-ATPase beta-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the beta-subunit influences the stability of the alpha beta complexes.
Calcium removal kinetics of the sarcoplasmic reticulum ATPase in skeletal muscle
Authors: Burmeister Getz E. E. , Lehman S. L.
Published: December 2017

The models of the sarcoplasmic reticulum (SR) Ca pump used to simulate Ca kinetics in muscle fibers are simple but inconsistent with data on Ca binding or steady-state uptake. We develop a model of the SR pump that is consistent with data on transient and steady-state Ca removal and has rate constants identified under near-physiological conditions. We also develop models of the other main Ca-binding proteins in skeletal muscle: troponin C and parvalbumin. These models are used to simulate Ca transients in cut fibers during and after depolarizing pulses. Simulations using the full SR pump model are contrasted with simulations using a Michaelis-Menten (MM) approximation to SR pump kinetics. The MM pump underestimates the amount of Ca released during depolarization, underestimates the initial rate of Ca binding by the pump, and overestimates the later rate of Ca pumping. These errors are due to fast initial binding by the SR pump, which is neglected in the MM approximation.
Purinergic-mediated inhibition of Na+-K+-ATPase in proximal tubule cells: elevated cytosolic Ca2+ is not required
Authors: Jin W. , Hopfer U.
Published: December 2017

The involvement of cytosolic Ca2+ concentration ([Ca2+]i) as messenger for the regulation of Na+-K+-ATPase activity was investigated in a renal cell line recently developed by immortalization of early proximal tubule primary cultures from the Wistar-Kyoto rat strain. Na+-K+-ATPase was measured as short-circuit current (Isc) in intact monolayers after permeabilization of the apical plasma membrane with amphotericin B. With symmetrical solutions, Isc quantitatively reflects Na+-K+-ATPase activity as judged by ouabain inhibition and dependence on Na+ and K+. Extracellular ATP (50% effective concentration = 0.32 mM) on the apical side produced acute inhibition of Na+-K+-ATPase-generated Isc of up to 50%. The inhibition peaked within 1 min and lasted approximately 5 min. The potency order was ATP > ADP >> beta,gamma-methyleneadenosine 5'-triphosphate = UTP, consistent with a P2y receptor. Extracellular ATP also stimulated a transient increase in [Ca2+]i. This increase had a similar time course as the inhibition of ATPase and reached a peak change of approximately 120 nM. However, the elevation of [Ca2+]i is not required in the purinergic inhibition of the Na+-K+-ATPase, since, first, increases in [Ca2+]i produced with a Ca2+ ionophore (ionomycin) failed to mimic the purinergic inhibition and, second, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, which abolished the [Ca2+]i elevation, failed to block the purinergic inhibition.
Leukotriene B4 costimulates 5-lipoxygenase activity in neutrophils via increased 5-lipoxygenase translocation
Authors: Serio K. J. , Baker J. R. , Ring W. L. , Riddick C. A. , Bigby T. D.
Published: December 2017

The goal of this investigation was to assess the effect of leukotriene B4 (LTB4) on 5-lipoxygenase activity and to examine the possible mechanisms of this effect. Exogenous LTB4 significantly increased the release of endogenous LTB4 from A-23187-stimulated neutrophils. The 5-lipoxygenase product release from A-23187-stimulated neutrophils decreased in the presence of an LTB4 receptor antagonist, suggesting that LTB4 has a receptor-mediated, autocrine effect on 5-lipoxygenase activity. Neutrophil 5-lipoxygenase activity increased significantly as cell density increased. In the presence of exogenous LTB4, no significant change in [14C]arachidonic acid release from neutrophils was observed. Exogenous LTB4 increased the amount of immunoreactive 5-lipoxygenase protein detected in the nuclear fraction of disrupted cells. LTB4 receptor antagonism decreased the amount of immunoreactive 5-lipoxygenase detected in the nuclear fraction. Thus LTB4 exerts an autocrine, receptor-mediated, costimulatory effect on 5-lipoxygenase activity. This feedback appears to have biological significance and involves enhanced 5-lipoxygenase translocation to the nuclear membrane.
Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel
Authors: Ding C. , Potter E. D. , Qiu W. , Coon S. L. , Levine M. A. , Guggino S. E.
Published: December 2017

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.
Inhibition of glucose transport in human red blood cells by adenosine antagonists
Authors: Gabel S. A. , O'Connell T. M. , Murphy E. , London R. E.
Published: December 2017

Previous studies have suggested that adenosine antagonists can interfere with normal glucose uptake in perfused rat heart. In the present studies, fluorine-19 nuclear magnetic resonance spectroscopy was used to study the effect of the adenosine antagonist, BW-A1433U, on the equilibrium exchange of fluorinated glucose analogs in human erythrocytes. Studies of the equilibrium exchange of both 2-fluoro-2-deoxy-D-glucose and 3-fluoro-3-deoxy-D-glucose with either one-dimensional magnetization transfer or two-dimensional exchange spectroscopy were performed, and significant inhibition was observed in all cases. From concentration-dependent studies, an inhibition constant for the equilibrium exchange measured at 37 degrees C of 24 microM was determined.
Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle
Authors: Nori A. , Nadalini K. A. , Martini A. , Rizzuto R. , Villa A. , Volpe P.
Published: December 2017

Calsequestrin (CS) is the junctional sarcoplasmic reticulum (jSR) Ca2+ binding protein responsible for intraluminal Ca2+ storage. The targeting mechanisms of CS to the jSR are yet to be unraveled. The nine-amino acid epitope of the influenza virus hemoagglutinin (referred to as HA1) was added at the COOH-terminal of CS by polymerase chain reaction cloning. The HA1-tagged CS cDNA was transiently transfected in either HeLa cells, myogenic cell lines, such as C2 and L8 cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of chimeric CS-HA1 were monitored by epifluorescence and confocal microscopy using either anti-CS antibodies or anti-HA1 antibodies. About 30% of transfected HeLa cells and 20-40% of myogenic cells expressed CS-HA1 into intracellular compartments, such as the perinuclear cisternae of endoplasmic reticulum (ER). Myoblasts of newborn rat skeletal muscles were first transfected and subsequently stimulated to differentiate into myotubes. CS-HA1 was detected in approximately 20% of transfected myotubes and did not affect CS distribution in myotubes. In the soleus muscle of adult rat, intramuscular injection of bupivacaine induced necrosis followed by regeneration. In vivo transfection of HA1-tagged CS cDNA in regenerating skeletal muscles determined expression in a few skeletal muscle fibers; CS-HA1 was localized only in jSR, as judged by confocal microscopy of longitudinal sections. The present results show that chimeric CS-HA1 is correctly sorted to ER/SR compartments and that the free COOH-terminal is not requested for sorting, retention, and segregation of CS to the SR.