Overexpression of human KCNA5 increases IK V and enhances apoptosis

Apoptotic cell shrinkage, an early hallmark of apoptosis, is regulated by K⁺ efflux and K⁺ channel activity. Inhibited apoptosis and downregulated K⁺ channels in pulmonary artery smooth muscle cells (PASMC) have been implicated in development of pulmonary vascular medial hypertrophy and pulmonary hypertension. The objective of this study was to test the hypothesis that overexpression of KCNA5, which encodes a delayed-rectifier voltage-gated K⁺ (Kv) channel, increases K⁺ currents and enhances apoptosis. Transient transfection of KCNA5 caused 25- to 34-fold increase in KCNA5 channel protein level and 24- to 29-fold increase in Kv channel current (I_K(V)) at +60 mV in COS-7 and rat PASMC, respectively. In KCNA5-transfected COS-7 cells, staurosporine (ST)-mediated increases in caspase-3 activity and the percentage of cells undergoing apoptosis were both enhanced, whereas basal apoptosis (without ST stimulation) was unchanged compared with cells transfected with an empty vector. In rat PASMC, however, transfection of KCNA5 alone caused marked increase in basal apoptosis, in addition to enhancing ST-mediated apoptosis. Furthermore, ST-induced apoptotic cell shrinkage was significantly accelerated in COS-7 cells and rat PASMC transfected with KCNA5, and blockade of KCNA5 channels with 4-aminopyridine (4-AP) reduced K⁺ currents through KCNA5 channels and inhibited ST-induced apoptosis in KCNA5-transfected COS-7 cells. Overexpression of the human KCNA5 gene increases K⁺ currents (i.e., K⁺ efflux or loss), accelerates apoptotic volume decrease (AVD), increases caspase-3 activity, and induces apoptosis. Induction of apoptosis in PASMC by KCNA5 gene transfer may serve as an important strategy for preventing the progression of pulmonary vascular wall thickening and for treating patients with idiopathic pulmonary arterial hypertension (IPAH). potassium ion channel; pulmonary hypertension     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Isolation and Characterization of the Olfactory Epithelial Cells of the Catfish

A preparation enriched in olfactory receptor cells has beenobtained from the olfactory mucosa of the catfish (Ictaluruspunctatus). The tissue was treated successively with trypsin,DNase, trypsin inhibitor, EDTA in Ca^{+ +} , Mg^{+ +} free mediumaccording to a method derived from that of Cohen, et al.⁽¹⁾.After mechanical disruption of the isolated olfactory lamellae,the cells were isolated by centrifugation on a Ficoll gradient.Each type of cell was morphologically identified by comparingin situ and in vitro preparations by SEM. Small round cellswere collected on 10% Ficoll. The nature of these cells is notknown but part of them are certainly basal cells which havebeen shown⁽²⁾ to be the precursors of the constantly regeneratingolfactory neurons. Respiratory cells settled mainly on 20% Ficoll.A fraction containing 60% sustentacular cells was collectedon 33% Ficoll. Olfactory cells characterized by an axon, a dendriteand several cilia, were found on 37% Ficoll. This fraction alsocontains up to 40% sustentacular cells. A yield of 20% was measuredfor olfactory cell isolation. Vital staining and ability tosynthesize RNA indicate a viability of the final preparationof 70% to 80%. Further identification of the cells was performedby measuring the binding activity of a series of amino acidsto a preparation enriched in olfactory cells. A good correlationwas determined between the extent of the binding and the reportedelectrophysiological activities of these amino acids recordedin vivo. Although the final olfactory cell suspension is notpure, it constitutes the first step in the study of the olfactoryreceptor sites.     

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22^phox and gp91^phox, as well as the Ca²⁺ concentration-responsive gp91^phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22^phox was not detectable, both gp91^phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22^phox or gp91^phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death. superoxide anion; diphenylene iodonium; p22^phox; gp91^phox; adenosine 3',5'-cyclic monophosphate response element; caspases     

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of Nostoc muscorum

ATPase activity in the cell membrane of a salt-stressed cyanobacterium,Nostoc muscorum M-14, was examined cytochemically by three differentstaining protocols. Application of Hulstaert's method resultedin distinct precipitation of the reaction products of ATPaseinside the cell membrane exclusively. No reaction products wereformed when ATP was replaced by GTP or when dicyclohexylcarbodiimideor N-ethylmaleimide was present in the reaction mixture. Bycontrast, low levels were detectable after the reaction in thepresence of ouabain. Bafilomycin did not affect the formationof products. Mayahara's method, which is considered to demonstratethe reaction of Na⁺,K⁺-ATPase activity, revealed the presenceof a ouabain-sensitive Na⁺,K⁺-ATPase in the cell membrane, whileWachstein-Meisel's method revealed the presence of an ATPaseactivity that was resistant to ouabain. It appears, therefore,that cell membranes of Nostoc muscorum contain both ouabain-sensitiveATPase and ouabain-insensitive ATPase. Comparison of the stainingprofiles of salt-stressed cells with those of control cellssuggested that a high-salt environment activates the ouabain-sensitiveNa⁺,K⁺-ATPase, which seems likely to be involved in the effluxof Na⁺ ions. (Received February 7, 1995; Accepted August 9, 1995)     

A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells

When Na-K-2Cl cotransport is activated in duckred blood cells by either osmotic cell shrinkage, norepinephrine,fluoride, or calyculin A, phosphorylation of the transporter occurs ata common set of serine/threonine sites. To examine the kinetics andregulation of the activating kinase, phosphatase activity was inhibitedabruptly with calyculin A and the subsequent changes in transporterphosphorylation and activity were determined. Increases in fractionalincorporation of ³²P into thetransporter and uptake of ⁸⁶Rb bythe cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in thismanner, the activating kinase was 1)stimulated by cell shrinkage, 2)inhibited by cell swelling, staurosporine, orN-ethylmaleimide, and3) unaffected by norepinephrine orfluoride. The inhibitory effect of swelling on kinase activity wasprogressively relieved by calyculin A, suggesting that the kinaseitself is switched on by phosphorylation. The kinetics of activation bycalyculin A conformed to an autocatalytic model in which thevolume-sensitive kinase is stimulated by a product of its own reaction(e.g., via autophosphorylation).

     

sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+ transport

To studythe role of sgk (serum, glucocorticoid-induced kinase) inhormonal regulation of Na⁺ transport mediated by theepithelial Na⁺ channel (ENaC), clonal cell lines stablyexpressing human sgk, an S422A sgk mutant, or aD222A sgk mutant were created in the background of the A6model renal epithelial cell line. Expression of normal sgkresults in a 3.5-fold enhancement of basal transport and potentiationof the natriferic response to antidiuretic hormone (ADH). Transfectionof a S422A mutant form of sgk, which cannot bephosphorylated by phosphatidylinositol-dependent kinase (PDK)-2, results in a cell line that is indistinguishable from the parent linein basal and hormone-stimulated Na⁺ transport. The D222Asgk mutant, which lacks kinase activity, functions as adominant-negative mutant inhibiting basal as well as peptide- andsteroid hormone-stimulated Na⁺ transport. Thussgk activity is necessary for ENaC-mediated Na⁺transport. Phosphorylation and activation by PDK-2 are necessary forsgk stimulation of ENaC. Expression of normal sgkover endogenous levels results in a potentiated natriferic response toADH, suggesting that the enzyme is a rate-limiting step for the hormoneresponse. In contrast, sgk does not appear to be therate-limiting step for the cellular response to aldosterone or insulin.

     

SYNCHRONIZATION OF BACTERIAL CULTURE BY PREINCUBATION OF CELLS AT HIGH CELL DENSITY

The growth of Escherichia coli strain B in a liquid medium wasfound to cease at a cell density of 5x10⁹ cells per ml. (Thiscritical concentration is designated as the maximum or M-concentration.)Even cells harvested from the logarithmic growth phase couldnot divide at this or higher cell densities. Investigationson the metabolic activities of such cultures, however, showedthat the synthesis of cellular protein and nucleic acid wastaking place under such circumstances, showing that only someprocess (or processes) particularly related to cell divisionwas suppressed at the critical cell concentration in question. This finding led us to devise a new method of synchronizationof E. coli: cells harvested from a logarithmic phase were preincubatedat the critical concentration of 5x10⁹ cells per ml for 45 minutes,and then diluted 100 times with fresh medium. This led to synchronizationof cell division, as shown by a stepwise multiplication in cellnumber. (Received June 20, 1961; )     

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. JayneBrahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl.Physiol. 83(3): 845-850, 1997.This study wasdesigned to investigate the effects of moderate-intensity endurancetraining on basal natural killer (NK) cell cytolytic activity in murinesplenocytes that were enriched for1)NK1.1⁺ cells or2) macrophages andNK1.1⁺ cells. Mice were assignedto sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn)groups. Splenocyte number, the percentages ofNK1.1⁺, large granular lymphocytes(NK1.1⁺, LGL-1⁺),and other subpopulations did not change in Trn mice. Approximately 70%of cells enriched for NK1.1⁺expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantlylower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remainedin the in vitro assay, LU perLGL-1⁺ cell did not differ acrossgroups. The results indicate that highly enrichedNK1.1⁺ cells from Trn mice hadlower NK cell activity compared with Sed mice. No differences in NKcell activity were observed when cells were enriched forNK1.1⁺ cells and macrophages.These findings support the hypothesis that macrophage modulation of NKcells may be one mechanism contributing to augmented basal NK cellactivity in endurance-trained individuals.

     

Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells

H⁺-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na⁺ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H⁺ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H⁺ activity and increased Na⁺ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H⁺-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na⁺ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na⁺ uptake and acid-base regulation mechanisms in zebrafish HR cells. ionocytes; Na⁺/H⁺ exchanger; skin; gill; embryo     

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

The possibility that protein kinase C (PKC) could control theactivity of L-type Ca²⁺ channelsin A7r5 vascular smooth muscle-derived cells in the absence of agoniststimulation was investigated using the patch-clamp technique.Consistent with the possibility that L-typeCa²⁺ channels are maximallyphosphorylated by PKC under these conditions, we show that1) activation of PKC with thephorbol ester phorbol 12,13-dibutyrate was ineffective in modulatingwhole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine orchelerythrine inhibited channel activity,3) inhibition of proteinphosphatases by intracellular dialysis of okadaic acid did not affectwhole cell currents, and 4) theinhibitory effect of staurosporine was absent in the presence ofokadaic acid. The inhibition ofCa²⁺ currents by PKC inhibitorswas due to a decrease in channel availability and long open events,whereas the voltage dependence of the open probability and thesingle-channel conductance were not affected. The evidence suggeststhat in resting, nonstimulated A7r5 cells there is a high level of PKCactivity that modulates the gating of L-typeCa²⁺ channels.

     

Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe

We examined the regulation of theNa⁺/H⁺exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinalC2/bbe cells, which spontaneously differentiate and have little basalapical NHE activity. Unidirectional apical membrane²²Na⁺influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of5-(N,N-dimethyl)amiloride-inhibitableflux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHEactivity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulatedby serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na⁺ influx), NHE2 and NHE3exhibited similar patterns of regulation, suggesting that the secondmessenger effects observed were not secondary to effects on cell pH.Thus, in contrast to their expression in nonepithelial cells, NHE2 andNHE3 expressed in an epithelial cell line behave similarly toendogenously expressed intestinal apical membrane NHEs. We concludethat physiological regulation and function of epithelium-specific NHEsare dependent on tissue-specific factors and/or conditionalrequirements.

     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

Arginine transport through system y(+)L in cultured human fibroblasts: normal phenotype of cells from LPI subjects

Inlysinuric protein intolerance (LPI), impaired transport of cationicamino acids in kidney and intestine is due to mutations of theSLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic aminoacid (CAA) transport in human fibroblasts obtained from LPI patientsand a normal subject. In both cell types the bidirectional fluxes ofarginine are due to the additive contributions of two Na⁺-independent, transstimulated transport systems. One ofthese mechanisms, inhibited by N-ethylmaleimide (NEM) andsensitive to the membrane potential, is identifiable with systemy⁺. The NEM- and potential-insensitive component,suppressed by L-leucine only in the presence ofNa⁺, is mostly due to the activity of systemy⁺L. The inward and outward activities of the two systemsare comparable in control and LPI fibroblasts. Both cell types expressSLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) aswell as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). Weconclude that LPI fibroblasts exhibit normal CAA transport throughsystem y⁺L, probably referable to the activity ofSLC7A6/y+LAT2.

     

Potassium channels in basolateral membrane vesicles from necturus enterocytes: stretch and ATP sensitivity

We have previously reported that ATP-inhibitable K⁺channels, in vesicles derived from the basolateral membrane ofNecturus maculosus small intestinal cells, exhibit volumeregulatory responses that resemble those found in the intact tissueafter exposure to anisotonic solutions. We now report that increases inK⁺ channel activity can also be elicited by exposure ofthese vesicles to isotonic solutions containing glucose or alanine thatequilibrate across these membranes. We also demonstrate that swellingafter exposure to a hypotonic solution or an isotonic solutioncontaining alanine or glucose reduces inhibition of channel activity byATP and that this finding cannot be simply attributed to dilution ofintravesicular ATP. We conclude that ATP-sensitive, stretch-activated K⁺ channels may be responsible for the well-establishedincrease in basolateral membrane K⁺ conductance ofNecturus small intestinal cells after the addition of sugarsor amino acids to the solution perfusing the mucosal surface, and wepropose that increases in cell volume, resulting in membrane stretch,decreases the sensitivity of these channels to ATP.

     

PLA2 stimulation of Na+/H+ antiport and proliferation in rat aortic smooth muscle cells

The proliferative properties and the ability to stimulate theNa⁺/H⁺antiport activity of a secretory phospholipaseA₂ were studied in rat aorticsmooth muscle cells in culture. The requirement of the enzymaticactivity of phospholipase A₂ toelicit mitogenesis was assessed by the use of ammodytin L, aSer⁴⁹ phospholipaseA₂ from the venom ofVipera ammodytes, devoid of hydrolyticactivity. We propose that the proliferative effect is mediated by thesame transduction pathway for both proteins. In particular,1) both secretory phospholipaseA₂ and ammodytin L stimulatedthymidine incorporation in a dose-dependent manner; 2) both proteins affected the cellcycle, as assessed by cell growth and fluorescence-activated cellsorting experiments; 3) bothphospholipase A₂ and ammodytin Lincreased intracellular pH, a permissive factor for cell proliferation,through activation of theNa⁺/H⁺antiport; 4) ammodytin L was able todisplace the ¹²⁵I-labeledphospholipase A₂ from specificbinding sites in a concentration range consistent with that capable ofeliciting a cellular response; and5) the inhibition by heparin wassimilar for both proteins, taking into account the ratio of heparin toprotein. In conclusion, the enzymatic activity of phospholipaseA₂ is not required for thestimulation of mitogenesis. The inhibitory effect of heparin combinedwith its therapeutic potential could help to clarify the role ofphospholipase A₂ in thepathogenesis of several preinflammatory situations.

     

Uptake and Metabolism of Sugars by Suspension Cultured Catharanthus roseus Cells

The uptake and metabolism of sugars by suspension-cultured Catharanthusroseus cells were investigated. Substantially all the sucrosein the culture medium was hydrolyzed to glucose and fructosebefore being taken up by the cells. The activity of invertasebound to cell walls, determined in situ, was high at the earlystage of culture. Glucose was more easily taken up by the cellsthan was fructose. Tracer experiments using [U-¹⁴C]glucose and[U-¹⁴C]fructose indicated that glucose is a better precursorfor respiration than fructose, while fructose is preferentiallyutilized for the synthesis of sucrose, especially in the earlyphase of cell growth. Possible metabolic routes of sugar insuspension-cultured Catharanthus roseus cells are discussedin the context of these results. Catharanthus roseus, Madagascar periwinkle, suspension culture, sucrose, glucose, fructose, metabolism, glycolysis     

Mechanisms of solute efflux from seed coats: whole-cell K+ currents in transfer cell protoplasts derived from coats of developing seeds of Vicia faba L.

In developing seed ofVicia faba L., solutes imported throughthe phloem of the coats move symplastically from the sieve elementsto a specialized set of cells (the thin-walled parenchyma transfercells) for release to the seed apoplast. Potassium (K⁺) is thepredominant cation released from the seed coats. To elucidatethe mechanisms of K⁺ efflux from seed coat to seed apoplast,whole-cell currents across the plasma membranes of protoplastsof thin-walled parenchyma transfer cells were measured usingthe whole-cell patch-clamp technique. Membrane depolarizationelicited a time-dependent and an instantaneous outward current.The reversal potential (E_R of the time-dependent outward currentwas close to the potassium equilibrium potential (E_K and itshifted in the same direction as E_K upon changing the externalK⁺ concentration, indicating that this current was largely carriedby an efflux of K⁺. The activation of the time-dependent outwardK⁺ current could be well fitted by two exponential componentsplus a constant. The instantaneous outward current could alsobe carried by K⁺ efflux as suggested by ion substitution experiments.These K⁺ outward rectifier currents elicited by membrane depolarizationare probably too small to represent the mechanism for the normalK⁺ efflux from seed coat cells. Membrane hyperpolarization morenegative than –80 mV activated a time-dependent inwardcurrent. K⁺ influx was responsible for the inward current asthe current reversed at membrane voltage close to E_K and shiftedin the same direction as E_K when external [K⁺] was varied. Activationof this K⁺inward rectifier current was well fitted with twoexponential components plus a constant. A regulating functionfor this current is suggested. Key words: Potassium outward rectifier, potassium inward rectifier, transfer cell protoplast, seed coat, Vicia faba L     

Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

Heme induces Cl^– secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl^– secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents (I_sc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced I_sc was caused by Cl^– secretion because it was inhibited in Cl^–-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl^– secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced I_sc. SnPP-induced I_sc was inhibited by the antioxidant vitamins, -tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl^– secretion can be regulated by the redox environment of the cell. heme oxygenase; electrolyte transport; carbon monoxide; cGMP; reactive oxygen species