Computational model of a circulation current that controls electrochemical properties in the mammalian cochlea

Sound-evoked mechanical stimuli permit endolymphatic K(+) to enter sensory hair cells. This transduction is sensitized by an endocochlear potential (EP) of +80 mV in endolymph. After depolarizing the cells, K(+) leaves hair cells in perilymph, and it is then circulated back to endolymph across the lateral cochlear wall. In theory, this process entails a continuous and unidirectional current carried by apical K(+) channels and basolateral K(+) uptake transporters in both the marginal cell and syncytial layers of the lateral wall. The transporters regulate intracellular and extracellular [K(+)], allowing the channels to form K(+) diffusion potentials across each of the two layers. These diffusion potentials govern the EP. What remains uncertain is whether these transport mechanisms accumulating across diverse cell layers make up a continuous circulation current in the lateral wall and how this current might affect the characteristics of the endolymph. To address this question, we developed an electrophysiological model that incorporates channels and transporters of the lateral wall and channels of hair cells that derive a circulation current. The simulation replicated normal experimental EP values and reproduced experimentally measured changes in the EP and intra- and extracellular [K(+)] in the lateral wall when different transporters and channels were blocked. The model predicts that, under these different conditions, the circulation current's contribution to the EP arises from different sources. Finally, our model also accurately simulated EP loss in a mouse model of a chloride channelopathy associated with deafness.     

Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the intrastrial fluid-blood barrier

The microenvironment of the cochlea is maintained by the barrier between the systemic circulation and the fluids inside the stria vascularis. However, the mechanisms that control the permeability of the intrastrial fluid-blood barrier remain largely unknown. The barrier comprises endothelial cells connected to each other by tight junctions and an underlying basement membrane. In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of perivascular cells with both macrophage and melanocyte characteristics. The perivascular-resident macrophage-like melanocytes (PVM/Ms) are in close contact with vessels through cytoplasmic processes. Here we demonstrate that PVM/Ms have an important role in maintaining the integrity of the intrastrial fluid-blood barrier and hearing function. Using a cell culture-based in vitro model and a genetically induced PVM/M-depleted animal model, we show that absence of PVM/Ms increases the permeability of the intrastrial fluid-blood barrier to both low- and high-molecular-weight tracers. The increased permeability is caused by decreased expression of pigment epithelial-derived factor, which regulates expression of several tight junction-associated proteins instrumental to barrier integrity. When tested for endocochlear potential and auditory brainstem response, PVM/M-depleted animals show substantial drop in endocochlear potential with accompanying hearing loss. Our results demonstrate a critical role for PVM/Ms in regulating the permeability of the intrastrial fluid-blood barrier for establishing a normal endocochlear potential hearing threshold.     

Potassium and activity of the sodium hydrogen exchanger isoform 1 in vascular smooth muscle of hypertensive rats.

Low potassium intake is inversely associated with blood pressure. In vitro, the proliferation of vascular smooth muscle cells (VSMCs) shows an inverse correlation with [K(+)]. In hypertension, many studies have established that the ubiquitous Na(+)/H(+) exchanger isoform 1 (NHE-1) exhibits increased activity and is permissive for cell proliferation. Changes in extracellular [K(+)] lead to altered intracellular Na(+) content, which could affect NHE activity and NHE-1 protein expression. We therefore investigated the effects of altering extracellular [K(+)] on NHE activity and NHE-1 expression in cultured VSMCs of both the spontaneously hypertensive rat (SHR) and its normotensive Wistar-Kyoto counterpart (WKY). Culture of SHR VSMCs for 48 hours in media containing 2, 4, 6, and 8 mmol. L(-1) [K(+)] led to activation of NHE-1 in the low [K(+)] media (NHE-1 activity at [K(+)] 2, 4, 6, and 8 mmol. L(-1) were 34.3 +/- 1.7, 29.5 +/- 1.1, 27.7 +/- 1.4, and 26.1 +/- 2.1 mmol. L(-1) min(-1), P <.006 by analysis of variance [ANOVA]). This was not associated with any significant changes in intracellular pH. By contrast, WKY VSMCs did not exhibit any significant activation of NHE-1 in low [K(+)] media (NHE-1 activity at [K(+)] 2, 4, 6, and 8 mmol. L(-1) were 24.3 +/- 2.9, 22.3 +/- 1.7, 19.0 +/- 1.8, and 18.6 +/- 1.6 mmol. l(-1) min(-1), P = not significant [NS] by ANOVA). Culture of SHR or WKY VSMCs in low [K(+)] media did not alter NHE-1 protein expression, suggesting the enhancement of activity in SHR cells was due to an increased turnover number of NHE-1. This response of NHE-1 in SHR VSMCs to K(+) depletion indicated a direct effect on these cells and could potentially enhance the contractile or proliferative phenotype of these cells in vivo.     

Influx of Calcium into Rabbit Myocardium in Relation to Its Ionic Environment

In the first 150 seconds after contracture of rabbit myocardium has been induced by shifting from perfusion with zero [Ca(2+)] and low [K(+)] to solutions with normal levels of those cations, there is a large influx of Ca(2+) as measured both by isotopic tracer flux and by total tissue [Ca]. Tracer studies indicate that the influx is 90 per cent complete in 90 seconds. Contracture due to substitution of either Li(+) or K(+) for Na(+) in perfusion fluids is also associated with an increased influx, but of lesser magnitude. The latter types of contracture are reversible while the former is not. It seems probable that the irreversible contracture is induced by the large Ca(2+) influx.     

Gastric parietal cell acid secretion in mice can be regulated independently of H/K ATPase endocytosis

BACKGROUND & AIMS: Gastric parietal cells secrete acid into the lumen of the stomach. They express a proton pump, the gastric H(+)/K(+) ATPase, the activity of which is tightly regulated. The H(+)/K(+) ATPase traffics between an intracytoplasmic compartment (tubulovesicles) in quiescent parietal cells and the apical plasma membrane in activated cells. These trafficking events are considered to contribute to the control of acid secretion by modulating access to apical K(+) and Cl(-) conductances that are required for transmembrane H(+) ion transport by the H(+)/K(+) ATPase. Here, we have determined whether the control of acid secretion in vivo requires membrane trafficking of the H(+)/K(+) ATPase. METHODS: We developed mice that only express an H(+)/K(+) ATPase beta subunit in which a putative tyrosine-based endocytosis motif in the cytoplasmic tail is mutated. Location of the H(+)/K(+) ATPase and parietal cell ultrastructure and gastric acid secretion were then examined. RESULTS: Parietal cells of these mice lacked a tubulovesicular compartment, and the H(+)/K(+) ATPase was resident exclusively on the apical plasma membrane. Despite the inability of the H(+)/K(+) ATPase to be endocytosed, the gastric acid secretory response to histamine or an antagonist was very similar to that of wild-type mice, indicating that control of H(+)/K(+) ATPase activity can occur independently of intracellular trafficking. CONCLUSIONS: We were able to dissociate the regulation of H(+)/K(+) ATPase activity from intracellular trafficking of the protein. Thus, it is likely that direct regulation of apical K(+) and Cl(-) conductances are sufficient to control gastric acid secretion.     

Effects of resveratrol, a grape polyphenol, on uterine contraction and Ca²+ mobilization in rats in vivo and in vitro

Dysmenorrhea is directly related to elevate prostaglandin F (PGF)(?α) levels. In Western medicine, this condition is treated using nonsteroidal antiinflammatory drugs. Because nonsteroidal antiinflammatory drugs produce many side effects, Chinese medicinal therapy is considered as a feasible alternative for treating dysmenorrhea. Many special physiological components used in Chinese medicine, such as resveratrol, have been isolated and identified. Resveratrol has many physiological functions, such as antioxidation and anticarcinogenic effects. However, the relationship between uterine smooth muscle contraction and resveratrol remains unknown. Here, we studied the in vitro and in vivo effects of resveratrol on uterine smooth muscle contraction. The uterus was separated from a female Sprague Dawley rat, and uterine smooth muscle contraction activity was measured and recorded. The results demonstrated that 1) resveratrol treatment inhibited PGF(?α)-, oxytocin-, acetylcholine-, and carbachol-induced uterine contractions in rats; 2) resveratrol inhibited uterine contractions stimulated by the Ca2(+) channel activator (Bay K 8644) and depolarization in response to high K(+) (KCl); 3) resveratrol inhibited PGF(?α)-induced increases in the [Ca2(+)]i in human uterine smooth muscle cells; 4) resveratrol could mimic Ca2(+) channel blockers to block Ca2(+) influx through voltage-operated Ca2(+) channels in the plasma membrane; and 5) resveratrol inhibited PGF(?α)-induced uterine contractions in rats in vivo. Resveratrol inhibited uterine contractions induced by PGF(?α) and high K(+) in a concentration-dependent manner in vitro; furthermore, it inhibited Ca2(+)-dependent uterine contractions. Thus, resveratrol consistently suppressed the increases in intracellular Ca2(+) concentrations ([Ca2(+)]i) induced by PGF(?α) and high K(+) concentrations. It can be assumed that resveratrol probably inhibited uterine contraction by blocking external Ca2(+) influx, leading to decreased [Ca2(+)]i. Therefore, resveratrol can be considered as a feasible alternative therapeutic agent for dysmenorrhea.     

Murine cerebral malaria: histopathology and ICAM 1 immunohistochemistry of the inner ear

Objective To evaluate the pathophysiologic changes in the inner ear during the course of severe cerebral malaria in an established animal model, C57 BL/6J mice. Methods This study aims to examine the hearing threshold, the histological changes and ICAM‐1 expression in the murine cochlea. Results Four of seven mice showed an expected hearing loss of 20 dB or more. The light microscopy of the inner ear did not show any morphologic alterations. The immunohistochemical analysis for ICAM‐1 showed intensive staining in the stria vascularis of sick animals and hardly any reaction in healthy controls. Conclusion The up‐regulation of ICAM‐1 in the stria vascularis – generating the endocochlear potential – suggests its involvement in plasmodial infection.     

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.     

Triggering mechanism for neurally mediated syncope induced by head-up tilt test: Role of catecholamines and response to propranolol

OBJECTIVESWe studied the triggering mechanism for neurally mediated syncope.BACKGROUNDAlthough increased transient sympathetic tone is thought to be necessary for the development of neurally mediated syncope, little is known about the triggering mechanism for neurally mediated syncope.METHODSPlasma epinephrine (EP) and norepinephrine (NE) levels were assessed in 20 syncope patients during tilt test (80°, 15 min) with and without isoproterenol (ISP, 0.01, 0.02 μg/kg/min). If syncope occurred, propranolol (0.1 mg/kg) was injected.RESULTSEight patients experienced syncope during tilting alone, and 9 patients required ISP for syncope. In the negative response without ISP, NE showed a small statistical 1.7-fold increase at end of tilting and EP did not change during tilting. When syncope occurred during tilting alone, a significant 11.7-fold increase in EP at syncope was registered concomitant with a small 2.5-fold increase in NE. When patients experienced syncope during tilting with ISP, a significant 5.0-fold increase in EP at syncope was registered concomitant with a small 1.7-fold increase in NE. In patients without ISP, propranolol did not interrupt syncope. In patients with ISP, six of eight receiving propranolol responded to tilting negatively.CONCLUSIONSAn increase of NE levels may result in inhibition of syncope and an EP surge may be a triggering mechanism for neurally mediated syncope. Comparatively low levels of EP may be enough to induce syncope during tilting with ISP compared with tilting alone. Propranolol is not effective in patients without ISP, but it frequently inhibits syncope in patients with ISP. Propranolol (0.1 mg/kg) may be insufficient to block the actions of high levels of circulating EP.     

Apico-basal inhomogeneity in distribution of ion channels in canine and human ventricular myocardium

OBJECTIVES: The aim of the present study was to compare the apico-basal distribution of ion currents and the underlying ion channel proteins in canine and human ventricular myocardium. METHODS: Ion currents and action potentials were recorded in canine cardiomyocytes, isolated from both apical and basal regions of the heart, using whole-cell voltage clamp techniques. Density of channel proteins in canine and human ventricular myocardium was determined by Western blotting. RESULTS: Action potential duration was shorter and the magnitude of phase-1 repolarization was significantly higher in apical than basal canine myocytes. No differences were observed in other parameters of the action potential or cell capacitance. Amplitude of the transient outward K(+) current (29.6+/-5.7 versus 16.5+/-4.4 pA/pF at +65 mV) and the slow component of the delayed rectifier K(+) current (5.61+/-0.43 versus 2.14+/-0.18 pA/pF at +50 mV) were significantly larger in apical than in basal myocytes. Densities of the inward rectifier K(+) current, rapid delayed rectifier K(+) current, and L-type Ca(2+) current were similar in myocytes of apical and basal origin. Apico-basal differences were found in the expression of only those channel proteins which are involved in mediation of the transient outward K(+) current and the slow delayed rectifier K(+) current: expression of Kv1.4, KChIP2, KvLQT1 and MinK was significantly higher in apical than in basal myocardium in both canine and human hearts. CONCLUSIONS: The results suggest that marked apico-basal electrical inhomogeneity exists in the canine-and probably in the human-ventricular myocardium, which may result in increased dispersion, and therefore, cannot be ignored when interpreting ECG recordings, pathological alterations, or drug effects.     

Aging cochleas in the F344 rat: morphological and functional changes

The Fischer 344 rat strain has been frequently used as an animal model of rapid aging. The present study was aimed at evaluating the incidence of apoptotic cells in the inner ear of 20-24-month-old F344 rats and to correlate it with cochlear function using otoacoustic emissions. Staining with cresyl violet and the enzymatic labeling (terminal deoxynucleotidyl transferase, TdT) of fragmented DNA revealed large numbers of apoptotic cells in the marginal and basal layers of the stria vascularis and in adjacent cells of the spiral ligament. The amplitudes of distortion products otoacoustic emissions (DPOAEs), which reflect functional state of the outer hair cells, were significantly reduced or totally absent in these animals. In contrast to old F344 rats, no marked DPOAE amplitude reduction and smaller numbers of apoptotic cells were found in young 4-month-old F344 rats or in aged 24-28-month-old Long Evans rats. The accumulation of apoptotic cells, mainly in the basal layer of the stria vascularis and in adjacent cells of the spiral ligament, leads to a detachment of the stria vascularis from the spiral ligament and results in the impairment of outer hair cell function. This specific type of strial deterioration suggests that aged F344 rats can serve as an animal model of strial presbycusis.     

Characterisation of the Na, K pump current in atrial cells from patients with and without chronic atrial fibrillation

OBJECTIVE: To assess the contribution of the Na, K pump current (I(p)) to the action potential duration (APD) and effective refractory period (ERP) in human atrial cells, and to investigate whether I(p) contributes to the changes in APD and ERP associated with chronic atrial fibrillation (AF). METHODS: Action potentials and ion currents were recorded by whole-cell patch clamp in atrial myocytes isolated from consenting patients undergoing cardiac surgery, who were in sinus rhythm (SR) or AF (>3 months). RESULTS: In cells from patients in SR, the I(p) blocker, ouabain (10 microM) significantly depolarised the membrane potential, V(m), from -80+/-2 (mean+/-S.E.) to -73+/-2 mV, and lengthened both the APD (174+/-17 vs. 197+/-23 ms at 90% repolarisation) and ERP (198+/-22 vs. 266+/-14 ms; P<0.05 for each, Student's t-test, n=7 cells, 5 patients). With an elevated pipette [Na(+)] of 30 mM, I(p) was measured by increasing extracellular [K(+)] ([K(+)](o)) from 0 to 5.4 mM. This produced an outward shift in holding current at -40 mV, abolished by 10 microM ouabain. K(+)- and ouabain-sensitive current densities were similar, at 0.99+/-0.13 and 1.12+/-0.11 pA/pF, respectively (P>0.05; n=9 cells), confirming the K(+)-induced current as I(p). I(p) increased linearly with increasing V(m) between -120 and +60 mV (n=25 cells). Stepwise increments in [K(+)](o) (between 0 and 10 mM) increased I(p) in a concentration-dependent manner (maximum response, E(max)=1.19+/-0.09 pA/pF; EC(50)=1.71+/-0.15 mM; n=27 cells, 9 patients). In cells from patients in AF, the sensitivity of I(p) to both V(m) and [K(+)](o) (E(max)=1.02+/-0.05 pA/pF, EC(50)=1.54+/-0.11 mM; n=44 cells, 9 patients) was not significantly different from that in cells from patients in SR. Within the group of patients in AF, long-term digoxin therapy (n=5 patients) was associated with a small, but significant, reduction in E(max) (0.92+/-0.07 pA/pF) and EC(50) (1.35+/-0.15 mM) compared with non-treatment (E(max)=1.13+/-0.08 pA/pF, EC(50)=1.76+/-0.14 mM; P<0.05 for each, n=4 patients). In cells from non-digoxin-treated patients in AF, the voltage- and [K(+)](o)-sensitivity (E(max) and EC(50)) were similar to those in cells from patients in SR. CONCLUSIONS: The Na, K pump current contributes to the human atrial cell V(m), action potential shape and ERP. However, the similarity in I(p) sensitivity to both [K(+)](o) and V(m) between atrial cells from patients with and without chronic AF indicates that I(p) is not involved in AF-induced electrophysiological remodelling in patients.     

Oncogenic Kit controls neoplastic mast cell growth through a Stat5/PI3-kinase signaling cascade

The D816V-mutated variant of Kit triggers multiple signaling pathways and is considered essential for malignant transformation in mast cell (MC) neoplasms. We here describe that constitutive activation of the Stat5-PI3K-Akt-cascade controls neoplastic MC development. Retrovirally transduced active Stat5 (cS5(F)) was found to trigger PI3K and Akt activation, and to transform murine bone marrow progenitors into tissue-infiltrating MCs. Primary neoplastic Kit D816V(+) MCs in patients with mastocytosis also displayed activated Stat5, which was found to localize to the cytoplasm and to form a signaling complex with PI3K, with consecutive Akt activation. Finally, the knock-down of either Stat5 or Akt activity resulted in growth inhibition of neoplastic Kit D816V(+) MCs. These data suggest that a downstream Stat5-PI3K-Akt signaling cascade is essential for Kit D816V-mediated growth and survival of neoplastic MCs.     

Cell membrane transport of magnesium

In order to maintain low intracellular Mg(2+) concentration ( [Mg(2+)] (i)), Mg(2+) has to be extruded from cell interior by active transport. In this article, properties of the active Mg(2+) transport in cardiac myocytes are reviewed. After [Mg(2+)] (i) was increased in the solution containing high Mg(2+) concentration, the reduction of extracellular Mg(2+) concentration ( [Mg(2+)] (o)) down to 1 mM caused a rapid decrease in [Mg(2+)] (i) only in the presence of extracellular Na(+);extracellular Na(+) stimulates the Mg(2+) efflux in a concentration dependent fashion with half maximal activation at 53 mM. When the rate of Mg(2+) efflux was compared under different levels of intracellular Na(+) concentration ( [Na(+)] (i)), intracellular Na(+) loading by ouabain decreased the rate of Mg(2+) efflux with 50% inhibition at - 40 mM [Na(+)] (i). In the experiments where the myocytes were voltage clamped at - 80 mV using the perforated patch-clamp technique with amphotericin B, the increase in pipette Na(+) concentration from 0 mM to 130 mM significantly decreased the rate of Mg(2+) efflux. The rate of Mg(2+) efflux was greater at the higher initial levels of [Mg(2 + )] (o), and was nearly zero at the basal levels;the efflux was half activated at 1.9 mM [Mg(2+)] (i). The Mg(2+) efflux was significantly slower at higher [Mg(2+)] (o) (50% inhibition at 10 mM). These results are consistent with the Mg(2+) efflux activity driven by the [Na(+)] gradient across cell membrane, or the Na(+)-Mg(2+) exchange.     

Altered growth response to prostaglandin E2 and its receptor signaling in mesangial cells from stroke-prone spontaneously hypertensive rats

OBJECTIVE: Prostaglandin (PG) E2, a major arachidonic acid metabolite in the kidney, acts on four receptor subtypes (EP1, EP2, EP3 and EP4). One of major causes of end-stage renal failure is hypertensive renal disease, in which enhanced renal PGE2 production has been shown. In this study, to explore the pathophysiological significance of EP subtypes in the kidney, we examined the role of EP subtypes on proliferation of mesangial cells (MCs) from stroke-prone spontaneously hypertensive rats (SHRSPs), which show faster growth than those from normotensive Wistar-Kyoto rats (WKYs). DESIGN AND METHODS: Using MCs from SHRSPs and WKYs, we investigated DNA synthesis and its upstream event, the phosphorylation of extracellular signal-regulated kinase (ERK), together with the gene expression of EP subtypes. RESULTS: Sulprostone, an EP1 agonist, dose-dependently increased DNA synthesis and the phosphorylation of ERK in MCs from both strains. The EP4 agonist, 11-deoxy-PGE1, inhibited sulprostone-induced phosphorylation of ERK in WKY-MCs. In contrast, 11-deoxy-PGE1 failed to inhibit the ERK activity in SHRSP-MCs. Interestingly, cAMP production mediated by EP4 was markedly attenuated in SHRSP-MCs as compared with that in WKY-MCs, despite the overproduction of endogenous PGE2 in SHRSP-MCs. Similar gene expressions of EP1 and EP4 and only faint expression of EP3 were detected in MCs from both strains. CONCLUSIONS: These results indicate that the PGE2/EP4 system counteracts the PGE2/EP1 system at the level of the intracellular signaling pathway. The altered EP4 signaling may play a critical role in the exaggerated mesangial growth in SHRSPs.     

Different thresholds of Notch signaling bias human precursor cells toward B-, NK-, monocytic/dendritic-, or T-cell lineage in thymus microenvironment

Notch receptors are involved in lineage decisions in multiple developmental scenarios, including hematopoiesis. Here, we treated hybrid human-mouse fetal thymus organ culture with the gamma-secretase inhibitor 7 (N-[N-(3,5-difluorophenyl)-l-alanyl]-S-phenyl-glycine t-butyl ester) (DAPT) to establish the role of Notch signaling in human hematopoietic lineage decisions. The effect of inhibition of Notch signaling was studied starting from cord blood CD34(+) or thymic CD34(+)CD1(-), CD34(+)CD1(+), or CD4ISP progenitors. Treatment of cord blood CD34(+) cells with low DAPT concentrations results in aberrant CD4ISP and CD4/CD8 double-positive (DP) thymocytes, which are negative for intracellular T-cell receptor beta (TCRbeta). On culture with intermediate and high DAPT concentrations, thymic CD34(+)CD1(-) cells still generate aberrant intracellular TCRbeta(-) DP cells that have undergone DJ but not VDJ recombination. Inhibition of Notch signaling shifts differentiation into non-T cells in a thymic microenvironment, depending on the starting progenitor cells: thymic CD34(+)CD1(+) cells do not generate non-T cells, thymic CD34(+)CD1(-) cells generate NK cells and monocytic/dendritic cells, and cord blood CD34(+)Lin(-) cells generate B, NK, and monocytic/dendritic cells in the presence of DAPT. Our data indicate that Notch signaling is crucial to direct human progenitor cells into the T-cell lineage, whereas it has a negative impact on B, NK, and monocytic/dendritic cell generation in a dose-dependent fashion.     

Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion.

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+, K(+)-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K(+)-ATPase--the so-called "19-kDa membranes"--containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the alpha chain, and a largely intact beta chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (approximately 9 kDa) unidentified peptide(s) are labeled by [14C]DCCD in a Rb(+)-protectable fashion. Rb(+)-protected [14C]DCCD incorporation into the "19 kDa membranes" and into native Na+,K(+)-ATPase is linearly correlated with inactivation of Rb+ occlusion. Similar linear correlations are observed when Rb(+)-protected [14C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per alpha chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the alpha chain. After cyanogen bromide digestion of purified, labeled alpha chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) approximately 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb(+)-protected [14C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.     

Biochemical localization of hepatic surface-membrane Na+,K+-ATPase activity depends on membrane lipid fluidity.

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.