In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

Caveolin-1 sensitizes cisplatin-induced lung cancer cell apoptosis via superoxide anion-dependent mechanism

Caveolin-1 (Cav-1) expression frequently found in lung cancer was linked with disease prognosis and progression. This study reveals for the first time that Cav-1 sensitizes cisplatin-induced lung carcinoma cell death by the mechanism involving oxidative stress modulation. We established stable Cav-1 overexpressed (H460/Cav-1) cells and investigated their cisplatin susceptibility in comparison with control-transfected cells and found that Cav-1 expression significantly enhanced cisplatin-mediated cell death. Results indicated that the different response to cisplatin between these cells was resulted from different level of superoxide anion induced by cisplatin. Inhibitory study revealed that superoxide anion inhibitor MnTBAP could inhibit cisplatin-mediated toxicity only in H460/Cav-1 cells while had no effect on H460 cells. Further, superoxide anion detected by DHE probe indicated that H460/Cav-1 cells generated significantly higher superoxide anion level in response to cisplatin than that of control cells. The role of Cav-1 in regulating cisplatin sensitivity was confirmed in shRNA-mediated Cav-1 down-regulated (H460/shCav-1) cells and the cells exhibited decreased cisplatin susceptibility and superoxide generation. In summary, these findings reveal novel aspects regarding role of Cav-1 in modulating oxidative stress induced by cisplatin, possibly providing new insights for cancer biology and cisplatin-based chemotherapy.     

Plasma membrane anion channels in higher plants and their putative functions in roots

Recent years have seen considerable progress in identifying anion channel activities in higher plant cells. This review outlines the functional properties of plasma membrane anion channels in plant cells and discusses their likely roles in root function. Plant anion channels can be grouped according to their voltage dependence and kinetics: (1) depolarization-activated anion channels which mediate either anion efflux (R and S types) or anion influx (outwardly rectifying type); (2) hyperpolarization-activated anion channels which mediate anion efflux, and (3) anion channels activated by light or membrane stretch. These types of anion channel are apparent in root cells where they may function in anion homeostasis, membrane stabilization, osmoregulation, boron tolerance and regulation of passive salt loading into the xylem vessels. In addition, roots possess anion channels exhibiting unique properties which are consistent with them having specialized functions in root physiology. Most notable are the organic anion selective channels, which are regulated by extracellular Al3+ or the phosphate status of the plant. Finally, although the molecular identities of plant anion channels remain elusive, the diverse electrophysiological properties of plant anion channels suggest that large and diverse multigene families probably encode these channels.     

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.     

Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions

In higher plants anion channels have recently been suggested to play key roles in controlling cellular functions, including turgor- and osmoregulation, stomatal movements, anion transport, signal transduction and possibly also signal propagation. In guard cells and roots, physiological functions of anion channels have been proposed which will be discussed here. In initial investigations it was proposed that anion channels in the plasma membrane of guard cells provide a prominent control mechanism for stomatal closing. The proposed model suggests that anion channel activation and the resulting anion efflux from guard cells cause membrane depolarization, thereby driving K⁺ efflux through outward-rectifying K⁺ channels required for stomatal closing. This article provides a brief review of new and recent insights into the molecular properties and cell biological functions of anion channels in guard cells. Furthermore, recently implicated putative functions of anion channels in roots during salt stress, xylem loading and Al³⁺ tolerance are addressed.     

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.     

Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C₆-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.     

Alternation of the slow with the quick anion conductance in whole guard cells effected by external malate

In previous investigations two anion conductances were discovered in guard-cell protoplasts: the quickly activating anion conductance (QUAC, R-type) and the slowly activating anion conductance (SLAC, S-type). In this investigation, effects of malate on the two anion conductances were tested in whole guard cells of Vicia faba L. by the use of the discontinuous single-electrode voltage-clamp method. Application of 1-s voltage ramps proved that QUAC displayed the malate shift of the activation threshold toward hyperpolarization also in complete guard cells. The sensitivity of SLAC to external malate was determined by responses to voltage pulses of 20 s duration at Cl- concentrations of 0.1, 3 or 50 mM. At no voltage were the currents measured at the end of the pulses in the presence and absence of malate significantly different from each other; the current-voltage relationship of SLAC appeared not to be affected by malate. However, in 32% of the cells exposed to malate, current activation in response to voltage steps occurred within 0.1 s, faster than was typical for SLAC, and activation was followed by inactivation with a half-time similar to 10 s: SLAC apparently had changed to QUAC. Simultaneously, the free-running membrane voltage depolarized at 0.1 mM Cl-, did not change at 3 mM Cl- and polarized at 50 mM Cl-, indicating that activation of QUAC increased the membrane conductance for anions and thereby drove the membrane voltage toward the equilibrium voltage of Cl-. The malate-induced changes were fully reversible at Cl- concentrations of 0.1 and 3 mM. These results reinforce the proposition that SLAC and QUAC represented two switching modes of the same anion channel (however, they do not suffice as proof); they also show that this interconvertibility can enable guard cells to control their membrane voltage rapidly.     

The ATP binding cassette transporter AtMRP5 modulates anion and calcium channel activities in Arabidopsis guard cells

Stomatal guard cells control CO(2) uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K(+) release from guard cells. Previous studies have shown that cytosolic Ca(2+) elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca(2+) activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca(2+)-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca(2+) signal transduction in guard cells.     

Anion channels in higher plants: functional characterization, molecular structure and physiological role

Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge. Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed. A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells. Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta.     

Anion channels: master switches of stress responses

During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure. Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.     

In vitro effects of different steroid hormones on superoxide anion production of human neutrophil granulocytes

Neutrophil granulocytes play an important role in atherogenesis also through their free radical generation. According to recent studies, a point of action by which estrogens can provide protection against atherosclerosis is their inhibiting effect on superoxide anion production. The aim of our study was to test whether this means a common effect of steroids on superoxide production, or whether various steroid hormones have different action on superoxide generation of human granulocytes. Neutrophils were separated from the blood samples of twelve healthy volunteers. Isolated cells were incubated with different concentrations (10(-9), 10(-8), 10(-7) M) of hydrocortisone, aldosterone, cortexolone, 17-beta-estradiol, progesterone, and testosterone. Superoxide anion production was determined by photometry using the reduction of ferricytochrome-C. Compared to that of control cells neutrophils incubated with 17-beta-estradiol, progesterone, testosterone and hydrocortisone showed significantly reduced superoxide production. No significant alteration of superoxide anion production was found after the incubation of cells with aldosterone and cortexolone. It is concluded that similarly to estradiol other sex steroids and cortisol can inhibit the free radical production of human granulocytes, but mineralocorticoid aldosterone and Reichstein's substance S do not show such activity. Our results provide new evidence supporting the theory that certain types of steroid hormones have antioxidant capacity. This may give further reasons for investigating the molecular background of the existence or absence of this property and thus might lead to the development of new free radical scavengers.     

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.     

Chemotactic peptide receptor-cytoskeletal interactions and functional correlations in differentiated HL-60 cells and human polymorphonuclear leukocytes

We studied the chemotactic peptide receptor/cytoskeletal interactions in HL-60 cells induced to differentiate with different agents and attempted to correlate these observations with the acquisition of different functional responses. Dibutyryl cyclic AMP-treated cells showed rapid superoxide anion production in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and slow, sustained response to phorbol myristate acetate (PMA). Retinoic acid-induced cells showed a slow, sustained response to both FMLP and PMA. Interferon-gamma-treated cells produced no superoxide anion on stimulation with FMLP, whereas tumor necrosis factor (TNF)-treated cells showed a slight response. Chemotactic peptide receptor association was the same in the HL-60 cells treated with different agents, despite marked differences in the superoxide anion generation and actin polymerization responses to FMLP and PMA in these cells. In mature neutrophils chemotactic peptide receptor association with the cytoskeleton was not affected by either pertussis or cholera toxin. However, both toxins inhibited FMLP-induced actin polymerization and superoxide anion generation. This suggested involvement of a G-protein similar to Gt, rather than Gi or Gs. Neither toxin had any effect on PMA-induced superoxide anion generation. These observations indicate that receptor association with the cytoskeleton may not have a significant role in affecting signal recognition and response. Among the several possible roles suggested, clearance of the occupied receptors may be the most important role of the cytoskeletal association. HL-60 cells induced to differentiate with different agents (because of their varied functional responses) might prove very useful in dissecting the molecular mechanisms regulating stimulus-induced activation of neutrophils.     

The reductive metabolism of nifurtimox and benznidazole in Crithidia fasciculata is similar to that in Trypanosoma cruzi

1. Addition of nifurtimox or benznidazole to the NADH-containing mitochondrial fraction of Crithidia fasciculata led to the appearance of the characteristic ESR spectra corresponding to their nitro anion radicals, suggesting that the nitro anion radical is a necessary intermediate in the reduction of both nitro compounds. 2. Nifurtimox anion radical generation by the mitochondrial fraction was insensitive to rotenone and antimycin A but was enhanced by KCN. 3. The nifurtimox anion radical reacted with oxygen under aerobic conditions leading to an increase in the cyanide-insensitive respiration of the intact cells and in the rate of O2- and H2O2 production by the C. fasciculata mitochondrial fraction. 4. In contrast, generation of O2- and H2O2 was not stimulated with pharmacological concentrations of benznidazole. Furthermore, benznidazole inhibited the cyanide-insensitive respiration of the intact cells.     

ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells.     

A new member of the HCO3(-) transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney

The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (alpha-type) and bicarbonate-secreting (beta-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H(+)-ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the alpha- and beta-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of beta-intercalated cells is the band 3 protein localized to the basolateral membrane of alpha-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of beta-intercalated cells. Cloned cDNA from the beta-intercalated cells shows about 30% homology with anion exchanger types 1-3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of beta-intercalated cells, although some beta-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the beta-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.     

Studies of the lungs in diabetes mellitus. II. Phospholipid analyses on the surfactant from broncho-alveolar lavage fluid of alloxan-induced diabetic rats

Structurally diverse anions (folate, 5-formyltetrahydrofolate, AMP, ADP, thiamine pyrophosphate, phosphate, sulfate, and chloride) that are competitive inhibitors of methotrexate influx in L1210 cells also enhance the efflux of methotrexate from these cells. The increase in efflux reaches a maximum of 2- to 4-fold depending upon the anion employed, and the anion concentrations required for half-maximal stimulation of efflux are similar to their K_i values for inhibition of methotrexate influx. A competitive inhibitor of methotrexate uptake (fluorescein-diaminopentane-methotrexate) that is not transported by this system, does not increase methotrexate efflux. These results suggest that the efflux of intracellular methotrexate is coupled to the concomitant uptake of an extracellular anion.