Atmospheric nitrogen deposition on petals enhances seed quality of the forest herb Anemone nemorosa.

Elevated atmospheric input of nitrogen (N) is currently affecting plant biodiversity and ecosystem functioning. The growth and survival of numerous plant species is known to respond strongly to N fertilisation. Yet, few studies have assessed the effects of N deposition on seed quality and reproductive performance, which is an important life-history stage of plants. Here we address this knowledge gap by assessing the effects of atmospheric N deposition on seed quality of the ancient forest herb Anemone nemorosa using two complementary approaches. By taking advantage of the wide spatiotemporal variation in N deposition rates in pan-European temperate and boreal forests over 2 years, we detected positive effects of N deposition on the N concentration (percentage N per unit seed mass, increased from 2.8% to 4.1%) and N content (total N mass per seed more than doubled) of A. nemorosa seeds. In a complementary experiment, we applied ammonium nitrate to aboveground plant tissues and the soil surface to determine whether dissolved N sources in precipitation could be incorporated into seeds. Although the addition of N to leaves and the soil surface had no effect, a concentrated N solution applied to petals during anthesis resulted in increased seed mass, seed N concentration and N content. Our results demonstrate that N deposition on the petals enhances bioaccumulation of N in the seeds of A. nemorosa. Enhanced atmospheric inputs of N can thus not only affect growth and population dynamics via root or canopy uptake, but can also influence seed quality and reproduction via intake through the inflorescences.

Revisiting CFTR inhibition: a comparative study of CFTRinh -172 and GlyH-101 inhibitors.

BACKGROUND AND PURPOSE: For decades, inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh -172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl(-) -conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non-CFTR-expressing cells. EXPERIMENTAL APPROACH: Using patch-clamp technique, we tested the effects of CFTRinh -172 and GlyH-101 inhibitors on three distinct types of Cl(-) currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl(-) conductance (VSORC) and finally the Ca(2+) -dependent Cl(-) conductance (CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines. KEY RESULTS: We confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl(-) conductance. However,GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh -172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations. CONCLUSIONS AND IMPLICATIONS: Both inhibitors affected Cl(-) conductances apart from CFTR. Our results provided insights into their use in mouse models.

Role of CFTR in oxidative stress and suicidal death of renal cells during cisplatin-induced nephrotoxicity.

The clinical use of the antineoplastic drug cisplatin is limited by its deleterious nephrotoxic side effect. Cisplatin-induced nephrotoxicity is associated with an increase in oxidative stress, leading ultimately to renal cell death and irreversible kidney dysfunction. Oxidative stress could be modified by the cystic fibrosis transmembrane conductance regulator protein (CFTR), a Cl(-) channel not only involved in chloride secretion but as well in glutathione (GSH) transport. Thus, we tested whether the inhibition of CFTR could protect against cisplatin-induced nephrotoxicity. Using a renal proximal cell line, we show that the specific inhibitor of CFTR, CFTR(inh)-172, prevents cisplatin-induced cell death and apoptosis by modulating the intracellular reactive oxygen species balance and the intracellular GSH concentration. This CFTR(inh)-172-mediated protective effect occurs without affecting cellular cisplatin uptake or the formation of platinum-DNA adducts. The protective effect of CFTR(inh)-172 in cisplatin-induced nephrotoxicity was also investigated in a rat model. Five days after receiving a single cisplatin injection (5 mg/kg), rats exhibited renal failure, as evidenced by the alteration of biochemical and functional parameters. Pretreatment of rats with CFTR(inh)-172 (1 mg/kg) prior to cisplatin injection significantly prevented these deleterious cisplatin-induced nephrotoxic effects. Finally, we demonstrate that CFTR(inh)-172 does not impair cisplatin-induced cell death in the cisplatin-sensitive A549 cancer cell line. In conclusion, the use of a specific inhibitor of CFTR may represent a novel therapeutic approach in the prevention of nephrotoxic side effects during cisplatin treatment without affecting its antitumor efficacy.

Weed seed bank response to 12 years of different fertilization systems.

Fertilizer amendments can impact weed populations in a variety of ways. This study evaluated the effects of 12 year-long applications of different fertilization systems on size and composition of the weed seed bank in a conventionally managed maize monoculture field. Fertilization systems included all factorial combinations of two dairy cattle slurry rates, three vegetable, fruit and garden waste (VFG) compost rates, and three synthetic N fertilizer rates. Soil samples were taken in each subplot in May 2008 after sowing and prior to herbicide application. Residues recovered from soil samples were tested for weed seedling emergence to characterize soil seed banks. Total weed seed bank density was affected by mineral N fertilization but not by compost or animal slurry application. Weed seed bank composition was related to compost amendment and mineral N fertilization. Annual compost amendments reduced seed bank density of some persistent species (e.g., Chenopodium album and Solanum nigrum) irrespective of mineral N fertilization. Compost is a promising tool for incorporation into integrated weed control strategies aimed at reducing weed seed bank persistence.

Cadmium causes delayed effects on renal function in the offspring of cadmium-contaminated pregnant female rats.

In the adult rat, chronic cadmium intoxication induces nephropathy with Fanconi-like features. This result raises the question of whether intoxication of pregnant rats has any deleterious effects on renal function in their offspring. To test this hypothesis, we measured the renal function of 2- to 60-day-old postnatal offspring from female rats administered cadmium chloride by the oral route (0.5 mg.kg(-1).day(-1)) throughout their entire gestation. Investigations of rat offspring from contaminated pregnant rats showed the presence of cadmium in the kidney at gestational day 20. After birth, the cadmium kidney concentration increased from postnatal day 2 to day 60 (PND2 to PND60), presumably because of 1) milk contamination and 2) neonatal liver cadmium content release. Although the renal parameters (glomerular filtration, U/P inulin, and urinary excretion rate) were not significantly affected until PND45, renal failure appeared at PND60, as demonstrated by a dramatic decrease of the glomerular filtration rate associated with increased excretion of the main ions. In parallel, an immunofluorescence study of tight-junction protein expression of PND60 offspring from contaminated rats showed a disorganization of the tight-junction proteins claudin-2 and claudin-5, specifically expressed in the proximal tubule and glomerulus, respectively. In contrast, expression of a distal claudin protein, claudin-3, was not affected. In conclusion, in utero exposure of cadmium leads to toxic renal effects in adult offspring. These results suggest that contamination of pregnant rats is a serious and critical hazard for renal function of their offspring.

Zinc protects renal function during cadmium intoxication in the rat.

This study investigates the effect in the rat of chronic CdCl2 intoxication (500 microg Cd2+/kg, daily i.p. injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2 (500 microg Zn2+/kg) during chronic CdCl2 intoxication. Our results indicate that 1) most of the filtered Cd2+ is reabsorbed within the kidney; 2) chronic Cd2+ intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2 exposure; 4) epithelial integrity is altered by chronic Cd2+ intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+ protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+ toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+ supplementation may provide a basis for future treatments.

Acute study of interaction among cadmium, calcium, and zinc transport along the rat nephron in vivo.

This study investigates the effect in rats of acute CdCl(2) (5 microM) intoxication on renal function and characterizes the transport of Ca(2+), Cd(2+), and Zn(2+) in the proximal tubule (PT), loop of Henle (LH), and terminal segments of the nephron (DT) using whole kidney clearance and nephron microinjection techniques. Acute Cd(2+) injection resulted in renal losses of Na(+), K(+), Ca(2+), Mg(2+), PO(4)(-2), and water, but the glomerular filtration rate remained stable. (45)Ca microinjections showed that Ca(2+) permeability in the DT was strongly inhibited by Cd(2+) (20 microM), Gd(3+) (100 microM), and La(3+) (1 mM), whereas nifedipine (20 microM) had no effect. (109)Cd and (65)Zn(2+) microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of (109)Cd were taken up. (109)Cd fluxes were inhibited by Gd(3+) (90 microM), Co(2+) (100 microM), and Fe(2+) (100 microM) in all nephron segments. Bumetanide (50 microM) only inhibited (109)Cd fluxes in LH; Zn(2+) (50 and 500 microM) inhibited transport of (109)Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute Cd(2+) intoxication are suggestive of proximal tubulopathy; 2) Cd(2+) inhibits Ca(2+) reabsorption possibly through the epithelial Ca(2+) channel in the DT, and this blockade could account for the hypercalciuria associated with Cd(2+) intoxication; 3) the PT is the major site of Cd(2+) reabsorption; 4) the paracellular pathway and DMT1 could be involved in Cd(2+) reabsorption along the LH; 5) DMT1 may be one of the major transporters of Cd(2+) in the DT; and 6) Zn(2+) is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1.

Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity.

Hypertonicity has pleiotropic effects on cell function, including activation of transporters and regulation of gene expression. It is important to investigate the action of hypertonicity on cystic fibrosis gene expression because cystic fibrosis transmembrane conductance regulator (CFTR), the cAMP-regulated Cl(-) channel, regulates ion transport across the secretory epithelia, which are often in a hypertonic environment. We found that adding >150 mosmol/l NaCl, urea, or mannitol to the culture medium reduced the amount of CFTR mRNA in colon-derived HT-29 cells in a time-dependent manner. Studies with inhibitors of various kinases [H-89 (protein kinase A inhibitor), bisindolylmaleimide (protein kinase C inhibitor), staurosporine (serine/threonine kinase inhibitor) and herbimycin A (tyrosine kinase inhibitor), SB-203580 and PD-098059 (mitogen-activated protein kinase inhibitors)] showed that CFTR gene expression and its decrease by added NaCl required p38 kinase cascade activity. The CFTR gene activity is regulated at the transcriptional level, since adding NaCl diminished the luciferase activity of HeLa cells transiently transfected with the CFTR promoter. This regulation requires protein synthesis. The complexity of the reactions involved in blocking CFTR gene transcription by NaCl strongly suggests that the decrease in CFTR mRNA is part of a general cell response to hyperosmolar stress.

Ammonium transport by the colonic H(+)-K(+)-ATPase expressed in Xenopus oocytes.

Functional expression of the rat colonic H(+)-K(+)-ATPase was obtained by coexpressing its catalytic alpha-subunit and the beta(1)-subunit of the Na(+)-K(+)-ATPase in Xenopus laevis oocytes. We observed that, in oocytes expressing the rat colonic H(+)-K(+)-ATPase but not in control oocytes (expressing beta(1) alone), NH(4)Cl induced a decrease in (86)Rb uptake and the initial rate of intracellular acidification induced by extracellular NH(4)Cl was enhanced, consistent with NH(+)(4) influx via the colonic H(+)-K(+)-ATPase. In the absence of extracellular K(+), only oocytes expressing the colonic H(+)-K(+)-ATPase were able to acidify an extracellular medium supplemented with NH(4)Cl. In the absence of extracellular K(+) and in the presence of extracellular NH(+)(4), intracellular Na(+) activity in oocytes expressing the colonic H(+)-K(+)-ATPase was lower than that in control oocytes. A kinetic analysis of (86)Rb uptake suggests that NH(+)(4) acts as a competitive inhibitor of the pump. Taken together, these results are consistent with NH(+)(4) competition for K(+) on the external site of the colonic H(+)-K(+)-ATPase and with NH(+)(4) transport mediated by this pump.

Aspirin and some other nonsteroidal anti-inflammatory drugs inhibit cystic fibrosis transmembrane conductance regulator protein gene expression in T-84 cells.

Cystic fibrosis (CF) is caused by mutations in the CF gene, which encodes CF transmembrane conductance regulator protein (CFTR), a transmembrane protein that acts as a cAMP-regulated chloride channel The disease is characterized by inflammation but the relationship between inflammation, abnormal transepithelial ion transport, and the clinical manifestations of CF are uncertain. The present study was undertaken to determine whether three nonsteroidal anti-inflammatory drugs (NSAIDs) (aspirin, ibuprofen, and indomethacin) modulate CFTR gene expression in T-84 cells. Treatment with NSAIDs reduced CFTR transcripts, and decreased cAMP-stimulated anion fluxes, an index of CFTR function. However, the two phenomena occurred at different concentrations of both drugs. The results indicate that NSAIDs can regulate both CFTR gene expression and the function of CFTR-related chloride transport, and suggest that NSAIDs act via multiple transduction pathways.

Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule.

1. Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca+2 concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT). 2. ATP raised [Ca2+]i in microdissected fura-2-loaded PCTs (half-maximal effect, approximately mumol 1(-1) ATP). The initial ATP-induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2-methylthioATP (2MeSATP) = ADP = ATP >> UTP, 2',3',-O-(4-benzoilbenzoil) ATP (BzATP), alpha, beta-methylene ATP (AMPCPP), adenosine. 3. In vivo electrophysiological measurements showed that 100 mumol 1(-1) peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl- solution, this ATP-induced depolarization was enhanced. These effects were inhibited by 1 mmol l-1 SITS, consistent with the activation of a basolateral Cl- conductance. 4. The ATP-induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin. 5. The ATP-induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporine and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content. 6. We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl- conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation.

Does the colonic H,K-ATPase also act as an Na,K-ATPase?

We previously have demonstrated that the colonic P-ATPase alpha subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H, K-ATPase would be Na+ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na+ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na+ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na+ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a beta subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na+ activity persists when diffusive Na+ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na+ channel, suggesting that the decrease is related to increased active Na+ efflux. The Na+ decrease depends on the presence of K+ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na+, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.

Symmetric pH dependence of buffering power in giant fused cells from frog kidney proximal tubule.

This study measures the intrinsic buffering power (beta(i)) of giant fused cells from the proximal kidney tubule of the frog (Rana ridibunda) as a function of intracellular pH (pHi). We monitored pHi and transmembrane potential difference during acid or alkaline cell loading, achieved by removal of NH4Cl-containing solutions or CO2-HCO3(-)-equilibrated solutions, respectively, in the absence of extracellular Na+. Data were well fit by the equation for a single, monoprotic buffer with a maximum beta(i) at a pHi of 7.39 +/- 0.06 and a total buffer concentration of 30.7 +/- 1.6 mM (means +/- SD). From pHi measurements obtained during CO2-HCO3- exposure, we also calculated the buffering power afforded by the CO2-HCO3- pair, and we show its increasing contribution to total buffering power at increasing PCO2 and pHi. To our knowledge, this is the first report of a cell type in which intrinsic cell buffers can be adequately approximated as a single monoprotic buffer with a negative logarithm of apparent dissociation constant in the normal physiological range and essentially symmetric dependence on pHi in both acid and alkaline ranges.

The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.

The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.

Further investigation of ionic diffusive properties and of NH4+ pathways in Xenopus laevis oocyte cell membrane.

To study the ionic diffusive properties and the NH4+ pathways in the Xenopus laevis oocyte cell membrane, we recorded the effects of various inhibitors on membrane potential (Vm) and membrane resistance (Rm); intracellular acidification was taken as an index of NH4+ influx from the bath to the cytoplasm. The following results were obtained: in the control state, barium and quinine (Q) ions depolarized Vm and raised Rm, consistent with inhibition of K+ conductance(s). Diphenylamine-2-carboxylic acid (DPC), 3',5'-dichlorodiphenylamine-2-carboxylic acid (DCDPC) and gadolinium ions hyperpolarized Vm and increased Rm, suggesting the inhibition of nonselective cationic conductance(s). In the presence of 20 mmol/l NH4Cl, Vm depolarized, Rm fell, and intracellular pH (pHi) decreased, consistent with an NH4+ influx. In the presence of DPC, the same manoeuvre induced a biphasic Vm change (i.e. a spike depolarization followed by a membrane hyperpolarization) and a fall of Rm; in most oocytes, intracellular acidification persisted and was reversible upon adding ouabain (Oua). These results indicate that a DPC-sensitive conductance is not the unique NH4+ pathway and that Na, K-ATPase may also mediate NH4+ influx. However, Oua did not prevent the Rm decrease, suggesting that ouabain-insensitive rheogenic pathway(s) are activated. Thus, we investigated the Vm change induced by NH4Cl addition in the presence of DPC: the spike depolarization followed by secondary hyperpolarization became a plateau depolarization when Q was added, suggesting involvement of Q-sensitive pathway(s) in the above described biphasic Vm change. In the presence of DPC, Q and Oua, intracellular acidification upon adding NH4Cl persisted consistent with further NH4+ influx through quinine-, DPC- and Oua-insensitive pathway(s).