Serum-induced inhibition of the phagocytic activity of cultured macrophages IC-21

Fetal bovine serum has been shown to considerably inhibit phagocytosis of non-opsonized 2-μm fluorescent latex beads by cultured macrophages IC-21. Phagocytic activity was assessed using fluorescent microscopy and specially devised ImageJ plugins. Phagocytosis percent, PP (percentage of the bead-containing cells in the cell population under study), and phagocytosis index, PI (mean number of beads per cell in the bead-containing population), were about 2 times lower in the cells incubated in the presence of 10% serum as compared to the respective parameters for the cells incubated in serum-free medium (55 ± 5% vs. 92 ± 1% and 2.0 ± 0.2 vs. 4.3 ± 0.2 beads/cell). The effect of serum was dose-dependent. Albumin (10 mg/ml) did not mimic the effect of serum, suggesting that fatty acid extraction was not the cause of the serum-induced inhibition. Serum is a source of exogenous cholesterol, therefore we checked if cholesterol removal could stimulate phagocytosis. Cholesterol-sequestering agent methyl-β-cyclodextrin (mβCD) in concentrations of 5–7 mM indeed caused an increase of the phagocytic activity but at 10 mM exerted an inhibitory effect in serum-free medium. Connexin channel blocker carbenoxolone (CBX, 250–500μM) in most cases inhibited phagocytosis; the presence of serum or mβCD modulated the CBX effects. The data indicate an important role of serum in regulation of the macrophage phagocytic activity. Stimulating effect produced by serum removal may partly be accounted for by a decrease of cholesterol concentration, which in turn may alter the functioning of integral proteins involved in the mechanisms of phagocytosis.     

Environmental KCl Causes an Upregulation of Apical Membrane Maxi K and ENaC Channels in Everted Ambystoma Collecting Tubule

Patch clamp methods were used to characterize the channels on the apical membrane of initial collecting ducts from Ambystoma tigrinum. Apical membranes were exposed by everting and perfusing fragments of the renal tubule in vitro. Tubules were dissected from two groups of animals; one maintained in tap water, and the other kept in a solution of 50 mm KCl from seven to nineteen days. Patches of apical membranes on tubules taken from animals exposed to tap water expressed low-conductance amiloride sensitive sodium channels (ENaC) in 22 of 49 patches. Only three maxi K channels were observed in this group. In animals exposed to KCl, low-conductance amiloride sensitive sodium channels, 3.7 ± 0.2 pS (36 of 45 patches) and high-conductance 98.3 ± 5.0 pS (19 of 45 patches) potassium channels were observed. The estimated density of apical maxi K channels increased dramatically from 0.08 to 0.76 channels/μ² in tubules taken from animals exposed to KCl. All but four of nineteen patches which contained maxi K channels also expressed the low conductance sodium channels. Therefore, at least 85% of the maxi K channels studied were in principal cells. We speculate that the increase in maxi K channel activity may represent a mechanism for enhancing the potassium secretory capacity of the initial collecting duct. As expected, exposure of the animals to 50 mm KCl prior to dissection of the initial collecting ducts also increased the estimated density of ENaC from 0.99 to 3.89 channels/μ². This upregulation of sodium channel activity is presumably related to the widely recognized effect of potassium loading to increase the plasma aldosterone level. Received: 25 August 1997/Revised: 13 November 1997     

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells

2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K⁺ outward currents. The IC ₅₀ value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BK_Ca channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BK_Ca channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BK_Ca channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BK_Ca channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Swelling-Activated K+ Efflux and Regulatory Volume Decrease Efficiency in Human Bronchial Epithelial Cells

This study describes the correlation between cell swelling-induced K⁺ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o⁻¹. Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T_c), as an index of cell volume, whereas ⁸⁶Rb efflux was assessed for K⁺ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H₂O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral ⁸⁶Rb efflux markedly increased during the hyposmotic shock, from 0.50 ± 0.03 min⁻¹ to a peak value of 6.32 ± 0.07 min⁻¹, while apical ⁸⁶Rb efflux was negligible. Channel blockers, such as GdCl₃ (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 μM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 μM) and genistein (150 μM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in ⁸⁶Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K⁺ and Cl⁻¹ efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral ⁸⁶Rb efflux. These data suggest that the basolateral extrusion of K⁺ and Cl⁻¹ from 16HBE14o⁻¹ cells in response to cell swelling determines RVD efficiency.     

ENaC Activity Requires CFTR Channel Function Independently of Phosphorylation in Sweat Duct

We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl⁻ conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na⁺ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl⁻ -channel function is required for activation. We used basilaterally α-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl⁻ -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl⁻ channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 μM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl⁻ current ( ) through CFTR was blocked by: 1) Cl⁻ removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 μM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous ΔF508 CF ducts). However, Cl⁻ gradients in the direction of absorption supported, while Cl⁻ gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated through CFTR in the direction of absorption (Cl⁻ gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing through CFTR in the direction of secretion (Cl⁻ gradient from cell to lumen) prevents ENaC activation even in the presence of through CFTR. An erratum to this article is available at .     

Characterization of the Na+/H+ Exchanger in the Luminal Membrane of the Distal Nephron

In the rabbit as well as the rat, a Na⁺/H⁺ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive ²²Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na⁺ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mm Na⁺ uptake was concentration dependent, with a IC₅₀ of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mm, the 35 mm Na⁺ uptake by the distal membrane was strongly inhibited by cimetidine (IC₅₀ 700 μm) and modestly inhibited by clonidine (IC₅₀ 1.6 mm). The incubation of proximal tubule suspensions with 1 mm (Bu₂) cAMP decreased the 15-sec EIPA-sensitive Na⁺ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1. Received: 6 March 1998/Revised: 6 July 1998     

A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

Basolateral K+ Conductance Establishes Driving Force for Cation Absorption by Outer Sulcus Epithelial Cells

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of −90.4 ± 0.7 mV (n= 78), close to the Nernst potential for K⁺ (−95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K⁺ concentration with a K⁺/Na⁺ permeability ratio of 36. The most frequently observed K⁺ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10⁻⁵ to 10⁻³ m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 μm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K⁺ conductance, in concert with the Na⁺,K⁺-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.     

X-ray microanalysis of elements in frozen-hydrated sections of an electrogenic K+ transport system: The posterior midgut of tobacco hornworm (Manduca sexta) in vivo andin vitro

Summary The lepidopteran midgut is a model for the oxygendependent, electrogenic K⁺ transport found in both alimentary and sensory tissues of many economically important insects. Structural and biochemical evidence places the K⁺ pump on the portasome-studded apical plasma membrane which borders the extracellular goblet cavity. However, electrochemical evidence implies that the goblet cell K⁺ concentration is less than 50mm. We used electron probe X-ray microanalysis of frozenhydrated cryosections to measure the concentration of Na, Mg, P, S, Cl, K, Ca and H₂O in several subcellular sites in the larval midgut ofManduca sexta under several experimental regimes. Na is undetectable at any site. K is at least 100mm in the cytoplasm of all cells. Typicalin vivo values (mm) for K were: blood, 25; goblet and columnar cytoplasm, 120; goblet cavity, 190; and gut lumen, 180. The high K concentration in the apically located goblet cavity declined by 100mm under anoxia. Both cavity and gut fluid are Cl deficient, but fixed negative charges may be present in the cavity. We conclude that the K⁺ pump is sited on the goblet cell apical membrane and that K⁺ follows a nonmixing pathway via only part of the goblet cell cytoplasm. The cavity appears to be electrically isolated in alimentary tissues, as it is in sensory sensilla, thereby allowing a PD exceeding 180 mV (lumen positive) to develop across the apical plasma membrane. This PD appears to couple K⁺ pump energy to nutrient absorption and pH regulation.     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

Ba2+-sensitive potassium permeability of the apical membrane in newt kidney proximal tubule

Summary The apical membrane K⁺ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V _a change) during alteration of luminal K⁺ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V _a change/decade for K⁺ was 50 mV at K⁺ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K⁺ concentration was increased from 2.5 to 25mm. Ba²⁺ (1mm in the lumen) reducedV _a change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba²⁺-sensitive K⁺ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K⁺ activity measured by K⁺-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K⁺ across both cell membranes. Thus, it is concluded that cell K⁺ passively diffuses, at least in part, through the K⁺ conductive pathway of the apical membrane.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH3 Cells

ATP-sensitive K⁺ (K_ATP) channels have been characterized in pituitary GH₃ cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 μm) revealed the presence of glibenclamide-sensitive K_ATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased K_ATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of K_ATP channels in a concentration-dependent manner with an IC₅₀ value of 30 μm. The activation of phospholipase A₂ caused by mellitin (1 μm) was found to enhance K_ATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced K_ATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the K_ATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that K_ATP channels similar to those seen in pancreatic β cells are functionally expressed in GH₃ cells. In addition to the presence of Ca²⁺-activated K⁺ channels, K_ATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential. Received: 19 June 2000/Revised: 13 September 2000     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Comparative study of K channel behavior inβ cell lines with different secretory responses to glucose

Summary The patch-clamp technique was used to identify and investigate two K channels in the cell membrane of the HIT cell, an insulin secreting cell line with glucose-sensitive secretion. Channel characteristics were compared with those of glucose-modulated K channels in the RINm5F cell, an insulin secreting cell line in which secretion is largely glucose insensitive. A 65.7 pS channel, identified with the ATP-sensitive K channel was seen in HIT cell-attached patches. Channel activity was dose-dependently inhibited by glucose, by 50 and 100% at 450 m and 8mm glucose, respectively, similar to the values previously reported for RIN cells. In inside-out patches channel activity was 50% inhibited by 56 m ATP and completely blocked between 500 m and 1mm, again, similar to the values reported for RIN cells.As in RIN cells a second, considerably larger (184 pS), K channel was glucose sensitive; the glucose sensitivity was similar to that in RIN cells with 50 and 100% channel inhibition at 7.5 and 25mm, respectively. After patch excision the mean channel conductance increased from 184 to 215 pS. Under these conditions activity was strongly calcium dependent in the rangepCa 5–7, identifying this as a calcium- and voltage-dependent K (K(Ca,V)) channel; the calcium sensitivity was similar to that of the adult rat cell K(Ca,V) channel. In inside-out RIN cell patches, the large K channel was less abundant but displayed a similar conductance (223 pS). However, its calcium sensitivity was more than 10 times lower than in HIT cells, similar to that of the K(Ca,V) channel in the neonatal rat cell, which also displays a reduced secretory response to glucose. Based on these observations, it is proposed that the low calcium sensitivity of the K(Ca,V) channel may be causally associated with secretory deficiency in RIN cells and the immature secretory response of the neonatal cell.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na⁺]_i in these cells loaded with the Na⁺ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na⁺]_i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA₂ activity (10 μm AACOCF₃) obviated EGF-induced increases in [Na⁺]_i. In contrast, PGE₂ (10 μg/ml) and cAMP (2 mm) increased [Na⁺]_i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na⁺]_i. Similarly, EGF failed to increase [Na⁺]_i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na⁺]_i followed by a decline towards its baseline value. EGF-induced increases in [Na⁺]_i were unaltered by inhibition of K⁺ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA₂ activity. Their stimulation increases PGE₂ and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001