Vesicular transport and apotransferrin in intestinal iron absorption, as shown in the Caco-2 cell model

The potential roles of vesicular transport and apotransferrin (entering from the blood) in intestinal Fe absorption were investigated using Caco-2 cell monolayers with tight junctions in bicameral chambers as a model. As shown previously, addition of 39 microM apotransferrin (apoTf) to the basolateral fluid during absorption studies markedly stimulated overall transport of 1 microM (59)Fe from the apical to the basal chamber and stimulated its basolateral release from prelabeled cells, implicating endo- and exocytosis. Rates of transport more than doubled. Uptake was also stimulated, but only 20%. Specific inhibitors of aspects of vesicular trafficking were applied to determine their potential effects on uptake, retention, and basolateral (overall) transport of (59)Fe. Nocodazole and 5'-(4-fluorosulfonylbenzoyl)-adenosine each reduced uptake and basolateral transport up to 50%. Brefeldin A inhibited about 10%. Tyrphostin A8 (AG10) reduced uptake 35% but markedly stimulated basolateral efflux, particularly that dependent on apoTf. Cooling of cells to 4 degrees C (which causes depolymerization of microtubules and lowers energy availability) profoundly inhibited uptake and basolateral transfer of Fe (7- to 12-fold). Apical efflux (which was substantial) was not temperature affected. Our results support the involvement of apoTf cycling in intestinal Fe absorption and indicate that as much as half of the iron uses apoTf and non-apoTf-dependent vesicular pathways to cross the basolateral membrane and brush border of enterocytes.     

Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells

In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca²⁺]_i. Low concentrations of ATP (<10 µM) induced intracellular Ca²⁺ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca²⁺]_i rise and increased the exocytosis rate sevenfold. The contribution of Ca²⁺ or cAMP pathways to exocytosis was tested by using the Ca²⁺ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca²⁺]_i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca²⁺ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate     

Cu loading alters expression of non-IRE regulated, but not IRE regulated, Fe dependent proteins in HepG2 cells

This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using ¹²⁵I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein - iron responsive element regulation mechanism.     

Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells

Synaptotagmin I (Syt I),a low-affinity Ca²⁺-binding protein, is thought to serve asthe Ca²⁺ sensor in the release of neurotransmitter.However, functional studies on the calyx of Held synapse revealed thatthe rapid release of neurotransmitter requires only approximatelymicromolar [Ca²⁺], suggesting that Syt I may play a morecomplex role in determining the high-affinity Ca²⁺dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinityCa²⁺-dependent exocytic pathways and express Syt I. Usingpatch-clamp capacitance measurements to monitor secretion and the acuteantisense deletion of Syt I from differentiated cells, we have shownthat the rapid and the most Ca²⁺-sensitive pathway ofexocytosis in rat melanotrophs requires Syt I. Furthermore, stimulationof the Ca²⁺-dependent exocytosis by cytosol dialysis withsolutions containing 1 µM [Ca²⁺] was completelyabolished in the absence of Syt I. Similar results were obtained by thepreinjection of antibodies against the CAPS (Ca²⁺-dependentactivator protein for secretion) protein. These results indicate thatsynaptotagmin I and CAPS proteins increase the probability of vesiclefusion at low cytosolic [Ca²⁺].

     

Prion protein fragment 106-126 potentiates catecholamine secretion from PC-12 cells

The toxic actions of scrapie prion protein(PrP^sc) are poorly understood. We investigated the abilityof the toxic PrP^sc fragment 106-126 to interfere withevoked catecholamine secretion from PC-12 cells. Prion protein fragment106-126 (PrP106-126) caused a time- andconcentration-dependent augmentation of exocytosis due to the emergenceof a Ca²⁺ influx pathway resistant to Cd²⁺ butsensitive to other inorganic cations. In control cells, secretion wasdependent on Ca²⁺ influx through L- and N-typeCa²⁺ channels, but after exposure to PrP106-126,secretion was unaffected by N-type channel blockade. Instead, selectiveL-type channel blockade was as effective as Cd²⁺ insuppressing secretion. Patch-clamp recordings revealed no change intotal Ca²⁺ current density in PrP106-126-treated cellsor in the contribution to total current of L-type channels, but a smallCd²⁺-resistant current was found only inPrP106-126-treated cells. Thus PrP106-126 augments secretionby inducing a Cd²⁺-resistant Ca²⁺ influxpathway and alters coupling of native Ca²⁺ channels toexocytosis. These effects are likely contributory factors in the toxiccellular actions of PrP^sc.

     

Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl^– secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (I_sc: thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca²⁺-chelating agent BAPTA-AM (50 µM) abolished the increase in I_sc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I_sc was observed. In addition, basolateral treatment with the PGE₂ receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on I_sc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca²⁺-activated KCNN4 K⁺ channel, and the KCNQ1 K⁺ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl^– conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl^– secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl^– secretion requires activation of CFTR and at least two distinct K⁺ channels located in the basolateral membrane. cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP     

Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells.

The effects of brefeldin A (BFA) on transferrin (Tf) transcellular transport, Tf receptor (TfR) distribution, and TfR-mediated endocytosis in filter-grown Madin-Darby canine kidney (MDCK) cells were studied. BFA (1.6 micrograms/ml) markedly enhanced the transcytosis of 125I-labeled Tf (125I-Tf) in both apical-to-basal and basal-to-apical directions; yet, BFA did not enhance the transcytosis of either native horseradish peroxidase (HRP) or membrane-bound HRP-poly(L-lysine) conjugates. Furthermore, this enhanced transcytosis of 125I-Tf was abolished either by competition with excess unlabeled Tf or by incubation at temperatures less than or equal to 25 degrees C. In addition, BFA treatment to MDCK cells: (a) increased 125I-Tf specific binding to the apical membrane and decreased 125I-Tf specific binding to the basal membrane; (b) decreased TfR recycling at the basolateral membrane; (c) altered the apical/basolateral distribution of TfRs in favor of the apical side; and (d) markedly increased 59Fe extraction, but not transcytosis, from apically endocytosed 59Fe-loaded Tf. These effects are consistent with a model in which BFA alters the traffic pattern of internalized Tf by decreasing basolateral TfR recycling, while diverting the nonrecycled fraction to the apical side of the cell. Our results indicate that, unlike the reported inhibition of polymeric IgA transcytosis (Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627), BFA can enhance the transcytosis of Tf in MDCK cells. Thus, by altering the intracellular traffic of ligand-receptor complexes, BFA can elicit either a decrease or an increase in transcytosis depending on the nature of the intracellular receptor processing.     

The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.     

Spatiotemporal analysis of exocytosis in mouse parotid acinar cells

Exocrine cells of the digestive system are specialized to secrete protein and fluid in response to neuronal and/or hormonal input. Although morphologically similar, parotid and pancreatic acinar cells exhibit important functional divergence in Ca²⁺ signaling properties. To address whether there are fundamental differences in exocytotic release of digestive enzyme from exocrine cells of salivary gland versus pancreas, we applied electrophysiological and optical methods to investigate spatial and temporal characteristics of zymogen-containing secretory granule fusion at the single-acinar cell level by direct or agonist-induced Ca²⁺ and cAMP elevation. Temporally resolved membrane capacitance measurements revealed that two apparent phases of exocytosis were induced by Ca²⁺ elevation: a rapidly activated initial phase that could not be resolved as individual fusion events and a second phase that was activated after a delay, increased in a staircaselike fashion, was augmented by cAMP elevation, and likely reflected both sequential compound and multivesicular fusion of zymogen-containing granules. Optical measurements of exocytosis with time-differential imaging analysis revealed that zymogen granule fusion was induced after a minimum delay of 200 ms, occurred initially at apical and basolateral borders of acinar cells, and under strong stimulation proceeded from apical pole to deeper regions of the cell interior. Zymogen granule fusions appeared to coordinate subsequent fusions and produced persistent structures that generally lasted several minutes. In addition, parotid gland slices were used to assess secretory dynamics in a more physiological context. Parotid acinar cells were shown to exhibit both similar and divergent properties compared with the better-studied pancreatic acinar cell regarding spatial organization and kinetics of exocytotic fusion of zymogen granules. membrane capacitance; differential imaging; zymogen; gland slice; exocrine cells     

Ca(2+) influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis

Previous reports showed that cleavage of vesicle-associatedmembrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting -cells inhibited Ca²⁺-evoked insulinsecretion. In these reports, the solubleN-ethylmaleimide-sensitive factor attachment protein targetreceptor proteins might have formed complexes, which preclude fullaccessibility of the putative sites for neurotoxin cleavage. In thiswork, VAMP-2 and SNAP-25 were effectively cleaved before they formedtoxin-insensitive complexes by transient transfection of insulinoma HITor INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A(BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca²⁺ (glucose orKCl)-evoked insulin release proportionate to the transfectionefficiency (40-50%) and an accumulation of insulin granules. Withthe use of patch-clamp capacitance measurements, Ca²⁺-evoked exocytosis by membrane depolarization to 10mV was abolished by TeTx (6% of control) but only moderately inhibitedby BoNT/A (30% of control). Depolarization to 0 mV to maximizeCa²⁺ influx partially overcame BoNT/A (50% of control) butnot TeTx inhibition. Of note, cAMP activation potentiatedCa²⁺-evoked secretion by 129% in control cells but only55% in BoNT/A-transfected cells and had negligible effects inTeTx-transfected cells. These results indicate that, whereas VAMP-2 isabsolutely necessary for insulin exocytosis, the effects of SNAP-25depletion on exocytosis, perhaps on insulin granule pool priming ormobilization steps, could be partially reversed by higher levels ofCa²⁺ or cAMP potentiation.

     

Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I-Tf to BMECs grown on matrix-coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high-affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4 degrees C indicated that there was a large intracellular receptor pool comprising 85-90% of the total cellular receptors. Accumulation of Tf at 37 degrees C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min-1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf-59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I-Tf was released into the medium with a rate coefficient of 0.017 min-1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post-confluent cells, filter-grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid-phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf-TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

Hypoxia controls iron metabolism and glutamate secretion in retinal pigmented epithelial cells

Background

Blood-barrier systems are essential in controlling iron levels in organs such as the brain and eye, both of which experience hypoxia in pathological conditions. While hypoxia's effects on numerous iron regulatory and storage proteins have been studied, little is known about how hypoxia affects iron metabolism. Iron also controls glutamate production and secretion; therefore the effects of hypoxia on iron metabolism and glutamate secretion were studied in polarized retinal pigmented epithelial (RPE) cells.

Methods

Primary canine RPE were cultured in Millicells to create polarized cell cultures. Iron uptake and efflux were measured in hypoxic and normoxic conditions. RPE were loaded with ⁵⁹Fe-transferrin. Glutamate concentrations in the cell conditioned media were also measured.

Results

Hypoxia induced a large increase in iron efflux from RPE in the basolateral direction. Glutamate secretion occurred mainly in the basolateral direction which is away from the retina and out of the eye in vivo. Glutamate secretion was doubled under hypoxic conditions.

Conclusions

Hypoxia is known to induce oxidative damage. The current results show that iron, a key catalyst of free radical generation, is removed from RPE under hypoxic conditions which may help protect RPE from oxidative stress. Results obtained here indicate the importance of using polarized tight junctional cells as more physiologically relevant models for blood-barrier-like systems.

General significance

While the effects of hypoxia on iron efflux and glutamate secretion may be protective for RPE cells and retina, increased glutamate secretion in the brain could cause some of the damaging neurological effects seen in stroke.     

Light-Dependent Iron Transport into Isolated Barley Chloroplasts

Translocation studies of ⁵⁹Fe(III)-epihydroxymugineic acid inintact barley plants revealed that Fe transport from leaf veinsto mesophyll cells is light-regulated. Similarly, Fe absorptionstudies with isolated chloroplasts showed that the Fe influxis light-dependent whereas its efflux occurred in the dark. (Received October 16, 1996; Accepted November 18, 1996)     

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals

In luteal cells, prostaglandin (PG)F_2a mobilizes intracellular calcium concentration ([Ca]_i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]_i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]_i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (), PGF_2a, and calcium ionophore were shown to increase [Ca]_i and stimulate intracellular ROS. With calcium ionophore and PGF_2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm^–1 · s^–1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm^–1 · s^–1; n = 5, P < 0.05). PGF_2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF_2a-stimulated luteal cells may occur through other signaling pathways. luteolysis; apoptosis; self-referencing microelectrode     

Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells

Exocytic insertion of H⁺-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H⁺-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H⁺-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H⁺-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H⁺-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H⁺-ATPase. In a pull-down assay of H⁺-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H⁺-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H⁺-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H⁺-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H⁺-ATPase vesicles. soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion     

The Action of Divalent Zinc, Cadmium, Mercury, Copper and Lead on the Trans-Root Potential and H+, Efflux of Excised Roots

Kennedy, C. D. and Gonsalves, F. A. N. 1987. The action of divalentzinc, cadmium, mercury, copper and lead on the trans-root potentialand H⁺ efflux of excised roots.—J. exp. Bot. 38: 800–817. The action of Zn²⁺, Cd²⁺, Hg²⁺, Cu²⁺ and Pb²⁺ ions on the trans-rootpotential and H⁺ efflux of young excised maize roots has beenstudied. Micro-electrode implantations into root epidermal cellsconfirmed the root outer membranes as the major contributorin the trans-root potential changes. The effects of these ionson H⁺ efflux were studied over a period of time in a continuousflow cell apparatus, adequate controls allowing for transientinterference due to divalent cations at the pH probe. The addition of Zn²⁺, 5 to 100 µmol dm^–3, to thesolution bathing the roots reduces H⁺ efflux and depolarizesthe trans-root potential. However, in the presence of Mg²⁺,0?1 or 1?0 mmol dm^–3, not only is this depolarizationinhibited, but hyperpolarization is observed instead. Cd²⁺ affectstrans-root potential and H⁺ efflux at a much slower rate thanZn²⁺, suggesting a lower membrane permeability. Without Mg²⁺,Cd²⁺ hyperpolarizes the trans-root potential, but this is bettersustained in its presence. Hyperpolarization did not occur withHg²⁺, Cu²⁺ or Pb²⁺ whether or not Mg²⁺ was present Hg²⁺ andto a lesser extent Cu²⁺ are potent depolarizers of the trans-rootpotential and strongly inhibit H⁺ efflux. The maximum rates of depolarization observed in the absenceof Mg²⁺ increase in the order Cd PCMBS <<.lt; Zn Cu< Hg. This is similar to the relative maximum rates of H⁺inhibition, Pb Cd <<.lt; Zn < Cu < Hg, suggestingconsiderable differences in mode of action and/or membrane permeability.The lower membrane permeability of the sulphydryl reagent PCMBSapparently prevents ready access to the site(s) of action availableto Hg²⁺. The reductions in trans-root potential and H⁺ gradients inducedby this range of cations would be detrimental to the acquisitionof nutrients using these gradients as an energy source. In contrast,Zn^2+, , in the presence of adequate Mg²⁺, could be beneficialto nutrient uptake by maintaining a higher membrane potentialthan would occur in its absence. Possible modes of action for the observed effects are discussed. Key words: Trans-root potentials, H⁺ efflux, heavy metal ions     

Membrane Depolarization Induced by N-Acetylchitooligosaccharide Elicitor in Suspension-Cultured Rice Cells

N-acetylchitooIigosaccharides (fragments of chitin) elicit defenseresponses, including phytoalexin production, in suspension-culturedrice cells. They induced rapid and transient membrane depolarizationaccompanied by a transient increase in net CP^–-efflux.The membrane depolarization was not affected by anaerobiosisor azide, suggesting that the major part of the depolarizationwas mediated by ion channels, not by energy-dependent ion pumps.Depolarization was partly inhibited in the presence of Ca²⁺-or Cl^–-channel blockers and highly inhibited by depletionof Ca²⁺ in the extracellular medium. A calcium ionophore, A23187 [GenBank] ,caused a transient depolarization but not an increase in Cl^–efflux, while it did not inhibit the elic-itor-induced transientdepolarization and Cl^– efflux. These suggest that theinflux of Ca²⁺ from the extracellular space to the cytoplasmis necessary as an initial trigger but not sufficient for membranedepolarization, Cl^– efflux, and the following signalingprocesses. (Received November 2, 1996; Accepted May 12, 1997)     

SNAP-25 is essential for cortical granule exocytosis in mouse eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has beenshown to play an important role inCa²⁺-dependent exocytosis inneurons and endocrine cells. During fertilization, sperm-egg fusioninduces cytosolic Ca²⁺mobilization and subsequentlyCa²⁺-dependent cortical granule(CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25is involved in this process. In this study, we determined theexpression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 weredetected in metaphase II (MII) mouse eggs by RT-PCR and immunoblotanalysis, respectively. Next, to determine the function of SNAP-25, weevaluated the change in CG exocytosis with a membrane dye,tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjectionof a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25in MII eggs. Sperm-induced CG exocytosis was significantly inhibited inthe BoNT/A-treated eggs. The inhibition was attenuated by coinjectionof SNAP-25. These results suggest that SNAP-25 may be involved inCa²⁺-dependent CG exocytosisduring fertilization in mouse eggs.

     

Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger

Allosteric regulation by cytosolic Ca²⁺ of Na⁺/Ca²⁺ exchange activity in the Ca²⁺ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca²⁺ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca²⁺ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, (241–680), that operates constitutively; i.e., its activity does not require allosteric Ca²⁺ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) compared with nontransfected cells. During Ca²⁺ entry through store-operated Ca²⁺ channels, Ca²⁺ efflux by the wild-type exchanger became evident only after [Ca²⁺]_i approached 100–200 nM. A subsequent decline in [Ca²⁺]_i was observed, suggesting that the activation process was time dependent. In contrast, Ca²⁺ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca²⁺]_i, the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca²⁺]_i had returned to resting levels. We conclude that Ca²⁺ efflux activity by the wild-type exchanger is allosterically activated by Ca²⁺, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca²⁺]_i to resting levels. persistent calcium activation; store-operated channels; calcium transient