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The purpose of this paper was to investigate the bone regeneration effect of autologous adipose tissue mesenchymal stem cells (ATMSC) in a small animal model. Twelve Wistar rats were given bilateral critical-size defects in the mandible. The defects were filled with coralline hydroxyapatite alone or combined with autologous undifferentiated ATMSC obtained from the dorsal fat pad. Studies were conducted at three and six weeks. Descriptive histology and histomorphometry revealed a significant (p < 0.05) increased bone regeneration values in the cell-treated defects at both three and six weeks. ATMSC promoted the formation of new bone in the central areas of the defects and in the scaffold micropores, both in a higher state of maturation. Autologous undifferentiated ATMSC enhanced bony healing of mandibular critical-size defects in rats when implanted with a coralline hydroxyapatite scaffold.

Producción Científica, Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6–12 months was 72.7%–77.8% for CLET cases and 76.5%–85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.), Ministerio de Sanidad, Consumo y Bienestar Social (project SAS/2481/2009), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León (grant SAN 1178/200), Red de Terapia Celular TerCel (project RD12/0019/0036)

Tissue engineering combining cross-linked serum scaffolds with bone-derived mesenchymal stem cells has displayed excellent results for repair of maxillofacial bone defects in animal models, but it had not been tested in humans yet. We present here a pilot clinical trial using autologous bone-derived mesenchymal stem cells (H-MSV) grown in a serum cross-linked scaffold (BioMax) for treatment of maxillary cysts in 9 patients. Cells obtained from alveolar bone were seeded in the BioMax scaffold prepared from autologous serum, expanded under GMP conditions, and subjected to osteogenic differentiation for 3–4 weeks before application. Evolution of the cystic cavity was followed by computerized tomography (CT) for 7 months. There was no inflammation or other adverse effects, and the CT density of the cyst interior increased significantly after the treatment. The ratio of the CT values after/before treatment was (mean ± SE) 2.52 ± 0.45; in contrast, the density of the contralateral control area of spongy alveolar bone without treatment did not change (ratio after/before, 0.99 ± 0.14). In conclusion, cell therapy with BioMax could be considered as an alternative therapy for maxillary bone defects and other losses of bone substance. Further research with allogeneic cells would be useful for reducing costs and improving logistics., Financial support from the Red de Terapia Cellular (RD16/0011/0003 and RD16/0011/0022) of the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, and the Centro en Red de Medicina Regenerativa de Castilla y León is gratefully acknowledged. The clinical trial was supported by grant EC10-255 from the Ministerio de Sanidad y Política Social (to LMR) and grant PI10/01566 from the Fondo de Investigaciones Sanitarias (FIS Intrasalud), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (to AS). EU co-financed these grants through the European Regional Development Fund. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

The concept of stimulus–secretion coupling was born from experiments performed in chromaffincells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca2+) entry and this generates a transient elevation of the cytosolic Ca2+ concentration ([Ca2+]c) that triggers the exocytotic release of catecholamines. The control of the [Ca2+]csignal is complex and depends on various classes of plasmalemmal calcium channels, cytosoliccalcium buffers, the uptake and release of Ca2+ from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca2+ extrusion mechanisms, such as the plasma membrane Ca2+-stimulated ATPase, and the Na+/Ca2+ exchanger. Computation of the rates of Ca2+ fluxes between the different cell compartments support the proposal that the chromaffincell has developed functional calcium tetrads formed by calcium channels, cytosoliccalcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca2+ transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytoticresponses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca2+]c transients occurring at different cell compartments, and generated by redistribution and release of Ca2+ by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo–endocytotic activities with energy production and protein synthesis., The work of the author’s laboratories has been supported by the following institutions. To AGG: (1) SAF2010-21795, Ministerio de Ciencia e Innovación (MCINN); (2) RETICS RD06/0009, Instituto de Salud Carlos III (MICINN); (3) S-SAL-0275-2006, Comunidad Autónoma de Madrid; (4) NDG07/9 y NDG09/8, Agencia Laín Entralgo, Comunidad Autónoma de Madrid, Spain. (5) Fundación Teófilo Hernando. To JGS: grants from the EU-ERA-Net programme, the Spanish Ministerio de Ciencia e Innovación (MICINN; SAF2008- 03175-E and BFU2010-17379), the Instituto de Salud Carlos III (RD06/0010/0000) and the Junta de Castilla y León (gr175).

7 páginas, 5 figuras.-- El pdf es la versión post-print del artículo., STIM1 and Orai1 are the main players in capacitative calcium entry (CCE). STIM1 senses [Ca2+] inside the endoplasmic reticulum (ER) and, when it decreases, opens Orai1, a store-operated calcium channel (SOC) in the plasma membrane that promotes Ca2+ entry and increases cytosolic Ca2+. The final destination of the entering Ca2+ is the ER, which refills very efficiently (capacitatively) with it. We propose here that SERCA is the third element of CCE, to which is tightly coupled to favour rapid Ca2+ pumping from the high Ca2+ microdomains, generated at the SOC's mouth, to the ER. We find that, on depletion of the intracellular Ca2+ stores, SERCA co-localizes with STIM1 at puncta. Adequate coupling of CCE and ER Ca2+ pumping requires correct proportions of STIM1, Orai1 and SERCA. Overexpression of Orai1 decreased modestly Ca2+ entry, but produced a dramatic fall of Ca2+ uptake into ER, which was rescued by STIM1 co-expression or by increasing external Ca2+. In permeabilized cells, Ca2+ uptake into the ER was indistinguishable in the Orai1-expressing and in the control cells. We propose that excess Orai1 uncouples SERCA from Ca2+ entry in the intact cell by disturbing the fine topology of Ca2+ pumping complexes within the ER-plasma membrane junctions., This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICINN, BFI2007-60157 and SAF2009- 03175-E) and the Instituto de Salud Carlos III. IMM was supported by a fellowship from the Spanish MICINN.

Endomembranes modify the progression of the cytosolic Ca2+ wave and contribute to generate Ca2+ microdomains, both in the cytosol and inside the own organella. The concentration of Ca2+ in the cytosol ([Ca2+]C), the mitochondria ([Ca2+]M) and the nucleus ([Ca2+]N) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca2+ from the high [Ca2+]C microdomains generated during cell activation near Ca2+ channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca2+ signal to the bulk cytosol. This shaping of [Ca2+]C signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca2+]M signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca2+]C signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca2+]N signals could be generated by direct release of stored Ca2+ into the nucleoplasm. Ca2+ release could even be restricted to subnuclear domains. Putative Ca2+ stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca2+ into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain. © 2006 Elsevier Ltd. All rights reserved., This work was funded by grants from the Ministerio de Educación y Ciencia (BFU2004-02765/BFI and BFU2005-05464) and from Junta de Castilla y León (VA016A05). Pablo Chamero holded a predoctoral fellowship from the Basque government.

Functional expression of GFP-VR1 Ca2+ channel. Single-cell [Ca2+]c image kinetic analysis of short pulse-treated cells expressing GFP-VR1. Transiently transfected JHM12 cells were loaded with fura 4F and subjected to digital imaging fluorescence microscopy, and [Ca2+]c images were obtained as indicated under “Experimental Procedures.” The first color frame (GFP) corresponds to the green fluorescence image, and subsequent ratiometric images for [Ca2+]c were recorded every 5 s. Representative frames corresponding to the different [Ca2+]c peaks were taken at the indicated time points . [Ca2+]c profiles were averaged for the two cell populations (GFP-VR1+, n = 45; GFP-VR1−, n = 62) and represented at the bottom of the Fig. 2 In Jambrina et al.. Cells were superfused during the indicated periods with the different solutions containing the VR1 agonist capsaicin (0.1 μm, CAPS) and the HM1R agonist carbachol (50 μm, CBCh), in the presence or the absence (Ca2+0) of calcium in the external medium. The capsaicin-sensitive cells corresponded to cells expressing GFP-VR1, as assessed by superimposition of fluorescence images excited at 490 nm (to visualize GFP) and the ratiometric Ca2+ images (fura-4F, excited at 340 and 380 nm; see upper panels in Fig. 2 A of Jambrina et al.). Color code included in Fig. 2; Blue represents 0 calcium whereas red is high calcium., We address the specific role of cytoplasmic Ca2+ overload as a cell death trigger by expressing a receptor-operated specific Ca2+ channel, vanilloid receptor sub-type 1 (VR1), in Jurkat cells. Ca2+ uptake through the VR1 channel, but not capacitative Ca2+ influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca2+] rises, exposure of phosphatidylserine, and cell death. Ca2+ influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca2+induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca2+ influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis., This work was supported by grants from Ministerio de Ciencia y Tecnología/European Union and Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (FEDER, 1FD97-1725-C02-01 and C02-02) and Comisión Interministerial de Ciencia y Tecnología, Spain, Grant SAF2000-0118-C03-02).

El pdf del artículo es el manuscrito de autor., Mitochondria take up calcium during cell activation thus shaping Ca2+ signaling and exocytosis. In turn, Ca2+ uptake by mitochondria increases respiration and ATP synthesis. Targeted aequorins are excellent Ca2+ probes for subcellular analysis, but single-cell imaging has proven difficult. Here we combine virus-based expression of targeted aequorins with photon-counting imaging to resolve dynamics of the cytosolic, mitochondrial, and nuclear Ca2+ signals at the single-cell level in anterior pituitary cells. These cells exhibit spontaneous electric activity and cytosolic Ca2+oscillations that are responsible for basal secretion of pituitary hormones and are modulated by hypophysiotrophic factors. Aequorin reported spontaneous [Ca2+] oscillations in all the three compartments, bulk cytosol, nucleus, and mitochondria. Interestingly, a fraction of mitochondria underwent much larger [Ca2+] oscillations, which were driven by local high [Ca2+] domains generated by the spontaneous electric activity. These oscillations were large enough to stimulate respiration, providing the basis for local tune-up of mitochondrial function by the Ca2+ signal., This work was supported by the Spanish Dirección General de Enseñanza Superior (DGES; Grants PB97-0474, APC1999-011, and 1FD97-1725-C02-02). C. Villalobos and L. Núñez hold postdoctoral fellowships from the Spanish DGES, and P. Chamero holds a predoctoral fellowship from the Basque Government.

Changes in the free calcium concentration of the endoplasmic reticulum ([Ca2+]er) play a central role controlling cellular functions like contraction, secretion or neuronal signaling. We recently reported that recombinant aequorin targeted to the endoplasmic reticulum (ER) [Montero M., Brini M., Marsault R. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 1995; 14: 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FASEB J 1997; 11: 881-886] can be used to monitor selectively [Ca2+]er in intact HeLa cells. Here we have used a herpes simplex virus type 1 (HSV-1) based system to deliver targeted aequorin into a number of different cell types including both postmitotic primary cells (anterior pituitary cells, chromaffin cells and cerebellar neurons) and cell lines (HeLa, NIH3T3, GH3 and PC12 cells). Functional studies showed that the steady state lumenal [Ca2+]er ranged from around 300 microM in granule cells to 800 microM in GH3 cells. InsP3-coupled receptor stimulation with agonists like histamine (in HeLa, NIH3T3 and chromaffin cells), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing hormone (TRH, in GH3 cells) produced a very rapid decrease in lumenal [Ca2+]er. Caffeine caused a rapid Ca2+ depletion of the ER in chromaffin cells, but not in the other cell types. Depolarization by high K+ produced an immediate and reversible increase of [Ca2+]er in all the excitable cells (anterior pituitary, GH3, chromaffin cells and granule neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynamic measurements of [Ca2+]er in several primary non-dividing cells.

Emptying the intracellular Ca2+ stores by treatment with the endomembrane Ca2+-ATPase inhibitor thapsigargin activates capacitative Ca2+ entry (CCE). This can be evidenced in fura-2-loaded cells by an increase of [Ca2+]i or by an acceleration of Mn2+ entry. Micromolar concentrations of arachidonic acid inhibited CCE induced by treatment with thapsigargin in rat thymocytes and in human neutrophils. This inhibitory action was shared by other unsaturated fatty acids, but not by the saturated arachidic acid nor by arachidonic acid methyl ester. The effect was not due to metabolites derived from arachidonic acid since several non-metabolizable analogs were able to reproduce it. Phorbol dibutyrate (PDB) acted similarly, suggesting that the inhibitory effect could be mediated by activation of protein kinase C (PKC). However, whereas the inhibition of CCE by PDB was reversed by treatment with the PKC inhibitor staurosporin, the inhibition by arachidonic acid was not. We find that unsaturated fatty acids antagonized microsomal dealkylation of benzyl-resorufin, a cytochrome P450-mediated activity, with the same specificity profile as for inhibition of CCE. These results are consistent with previous proposals suggesting that a microsomal cytochrome P450 may be involved in the regulation of CCE.

Exposure of bovine chromaffin cells to 30 microM veratridine for 24 h led to 70-80% cell death as reflected by phase contrast microscopy, trypan blue exclusion, lactate dehydrogenase (LDH) release and cell catecholamine contents. Na+ deprivation, Ca2+ deletion or tetrodotoxin (5 microM) prevented the veratridine-induced cell damage. Nimodipine and verapamil, but not omega-conotoxin GVIA afforded 20-30% protection. Flunarizine protected the cells by 80% and R56865 by 60%. Stimulation of fura-2-loaded single bovine chromaffin cells with 30 microM of 1,1-dimethyl-4-phenylpiperazinium (DMPP) or 59 mM K+ caused fast increases in cytosolic Ca2+ concentrations, ([Ca2+]i). The [Ca2+]i rose from 0.1 to peaks of 1.9 microM, which quickly declined to near basal levels with a t1/2 of around 30 s. In spite of sustained stimulation with these two depolarizing agents, the [Ca2+]i remained low and did not undergo oscillations. In contrast, veratridine (30 microM) caused large and frequent oscillatory changes in the [Ca2+]i which were long-lasting and did not disappear even 30 min after washing out the toxin. The [Ca2+]i oscillations were reversibly suppressed by Na+ or Ca2+ removal and by 5 microM tetrodotoxin. Selective L-type Ca2+ channel blockers (10 microM nimodipine or verapamil) or N-type Ca2+ channel blockers (1 microM omega-conotoxin GVIA) did not affect the [Ca2+]i oscillations. In contrast, flunarizine or R56865 (10 microM each) suppressed the oscillations of [Ca2+]i. The results demonstrate that bovine chromaffin cells have the necessary machinery to develop prolonged and repetitive [Ca2+]i oscillations in the presence of veratridine; however, 'physiological' depolarizing stimuli did not cause oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of 1,4-dihydropyridine derivatives on divalent cation transients and catecholamine release stimulated by either high K+ or the nicotinic receptor agonist dimethyl-phenyl-piperazinium (DMPP) have been compared in bovine adrenal chromaffin cells. The activation of Ca2+ entry pathways was followed by measuring 45Ca2+ or Mn2+ uptake, or by the changes of [Ca2+]i in fura-2-loaded chromaffin cells. Various dihydropyridine Ca2+ channel blockers (nimodipine, PCA50938, nifedipine, nitrendipine, furnidipine) abolished the DMPP-mediated effects, but prevented only partially the activation by high [K+]0 of 45Ca2+ uptake. The IC50 for DMPP-induced activation was around 1 μM. The L-type Ca2+ channel activator Bay K 8644 potentiated the uptake of 45Ca+ induced by K++ depolarization at concentrations between 10 nM and 1 μM, but completely inhibited the uptake of 45Ca2+ by DMPP (IC50, 0.9 μM). Both high [K+]0 and DMPP produced membrane depolarization as measured using bis-oxonol. The DMPP-evoked, but not the K+-evoked membrane depolarization was prevented by Na+ removal, suggesting that the depolarization was due to Na+ entry through the acetylcholine receptor ionophore. Nimodipine at 10 μM abolished the depolarization induced by DMPP, leaving the K+-evoked depolarization unaffected. Tetrodotoxin (2 μM) did not affect the DMPP- or high K+-mediated cell depolarization. Whole-cell inward current evoked by 100 μM DMPP (IDMPP) was measured in cells voltage-clamped at −80 mV. Nimodipine (10 μM) reduced IDMPP by 36%; Bay K 8644 (10 μM) inhibited IDMPP by 67%. DMPP-evoked catecholamine release from superfused chromaffin cells was reduced by over 90% with 10 μM nimodipine; in contrast, K+-evoked release was decreased by 20%. The results suggest that the nicotinic acetylcholine receptor of bovine chromaffin cells contains a site for dihydropyridines whose occupation blocks Na+ entry through the receptor ionophore. This chromaffin cells contains a site for dihydropyridines whose occupation blocks Na+ entry through the receptor ionophre. This limits the ensuing membrane depolarization, firing of action potentials, recruitment of Ca2+ channels and entry of Ca2+ in the cells, leading to a decrease in catecholamine secretion.

The sensitivity to Ca 2+ of the Ca 2+ -dependent K + channel can be increased by the artificial electron donor system ascorbate + phenazine-methosulphate in a variety of animal cells. In the human erythrocyte the shift from the ‘low’ to the ‘high-affinity’ state seems to depend on the reduction of a membrane component accepting 2 electrons and with an standard redox potential (pH 7.5) of about 47 mV. The relevance of this redox modulation under physiological circumstances is unknown at the moment.

When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb + (K + ) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb + (K + ) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca 2+ (Gardos effect). The electron donors require the presence of intracellular Ca 2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K + . Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca 2+ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca 2+ more effective to elicit rapid K + movements. Plasma membrane oxidoreductase activities could be engaged in this change.

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.

The effects of variations of the NAD+/NADH quotient on the uptake of 86Rb by human red cells loaded by non-disruptive means with the chelator Benz2 and different amounts of 45Ca has been examined. The NAD+/NADH quotient was modified by the addition of pyruvate and/or lactate or xylitol. It was found that the uptake of 86Rb at a given intracellular Ca2+ concentrations was faster in the reduced state (lactate or xylitol added). Metabolic changes were associated with variations of the redox state. However, glycolitic intermediates did not significantly modify the apparent affinity for Ca2+ of the Ca2+-dependent K+ channel in one-step inside-out vesicles prepared from the erythrocyte membrane. Taken together, these results suggest that modifications of the cytoplasmic redox potential could modulate the sensitivity to Ca2+ of the Ca2+-dependent K+ channel in the human red cells under physiological conditions. This conclusion is consistent with previous findings in inside-out vesicles of human erythrocytes using artificial electron donors.

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K + permeability had been increased, either by valinomycin or by activation of Ca 2+ -dependent K + channels, in medium containing different K + concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na + -dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2–3.1-times for each 60 mV-hyperpolarization. The Na + -dependent system ASC was insensitive to membrane potential. The Na + -independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.

Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity.

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5·10−8 M) while oxidized cytochrome c decreases the sensitivity to about 1 3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted for by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.