Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes.

BACKGROUND: A concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency. METHODS: In 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers. RESULTS: Patients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate. CONCLUSION: The positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.

Serum calcium levels are associated with novel cardiometabolic risk factors in the population-based CoLaus study.

BACKGROUND: Associations of serum calcium levels with the metabolic syndrome and other novel cardio-metabolic risk factors not classically included in the metabolic syndrome, such as those involved in oxidative stress, are largely unexplored. We analyzed the association of albumin-corrected serum calcium levels with conventional and non-conventional cardio-metabolic risk factors in a general adult population. METHODOLOGY/PRINCIPAL FINDINGS: The CoLaus study is a population-based study including Caucasians from Lausanne, Switzerland. The metabolic syndrome was defined using the Adult Treatment Panel III criteria. Non-conventional cardio-metabolic risk factors considered included: fat mass, leptin, LDL particle size, apolipoprotein B, fasting insulin, adiponectin, ultrasensitive CRP, serum uric acid, homocysteine, and gamma-glutamyltransferase. We used adjusted standardized multivariable regression to compare the association of each cardio-metabolic risk factor with albumin-corrected serum calcium. We assessed associations of albumin-corrected serum calcium with the cumulative number of non-conventional cardio-metabolic risk factors. We analyzed 4,231 subjects aged 35 to 75 years. Corrected serum calcium increased with both the number of the metabolic syndrome components and the number of non-conventional cardio-metabolic risk factors, independently of the metabolic syndrome and BMI. Among conventional and non-conventional cardio-metabolic risk factors, the strongest positive associations were found for factors related to oxidative stress (uric acid, homocysteine and gamma-glutamyltransferase). Adiponectin had the strongest negative association with corrected serum calcium. CONCLUSIONS/SIGNIFICANCE: Serum calcium was associated with the metabolic syndrome and with non-conventional cardio-metabolic risk factors independently of the metabolic syndrome. Associations with uric acid, homocysteine and gamma-glutamyltransferase were the strongest. These novel findings suggest that serum calcium levels may be associated with cardiovascular risk via oxidative stress.

PTH and 1.25 vitamin D response to a low-calcium diet is associated with bone mineral density in renal stone formers.

BACKGROUND: Renal calcium stones and hypercalciuria are associated with a reduced bone mineral density (BMD). Therefore, the effect of changes in calcium homeostasis is of interest for both stones and bones. We hypothesized that the response of calciuria, parathyroid hormone (PTH) and 1.25 vitamin D to changes in dietary calcium might be related to BMD. METHODS: A single-centre prospective interventional study of 94 hyper- and non-hypercalciuric calcium stone formers consecutively retrieved from our stone clinic. The patients were investigated on a free-choice diet, a low-calcium diet, while fasting and after an oral calcium load. Patient groups were defined according to lumbar BMD (z-score) obtained by dual X-ray absorptiometry (group 1: z-score <-0.5, n = 30; group 2: z-score -0.5-0.5, n = 36; group 3: z-score >0.5, n = 28). The effect of the dietary interventions on calciuria, 1.25 vitamin D and PTH in relation to BMD was measured. RESULTS: An inverse relationship between BMD and calciuria was observed on all four calcium intakes (P = 0.009). On a free-choice diet, 1.25 vitamin D and PTH levels were identical in the three patient groups. However, the relative responses of 1.25 vitamin D and PTH to the low-calcium diet were opposite in the three groups with the highest increase of 1.25 vitamin D in group 1 and the lowest in group 3, whereas PTH increase was most pronounced in group 3 and least in group 1. CONCLUSION: Calcium stone formers with a low lumbar BMD exhibit a blunted response of PTH release and an apparently overshooting production of 1.25 vitamin D following a low-calcium diet.

Calcium-sensing receptors modulate renin release in vivo and in vitro in the rat.

OBJECTIVES: Calcium-sensing receptors (CaSRs) have been localized in the juxtaglomerular apparatus where they may contribute to the regulation of renin release. In the present study, we investigated the in-vitro and in-vivo effects of the calcimimetic R-568 on renin release. METHODS: In vitro, the effect of calcimimetics on renin release was assessed by incubating freshly isolated rat juxtaglomerular cells with or without R-568 (1 and 10 mumol/l) in serum-free medium in the presence or absence of forskolin or CaCl2. In vivo, we measured the impact of R-568 (20 ng/min intravenously) on the acute changes in plasma renin activity (PRA) induced by either a 90 min infusion of the angiotensin-converting enzyme inhibitor captopril, or the beta-receptor agonist isoproterenol, or of a vehicle in or after a furosemide challenge in conscious Wistar rats. RESULTS: In vitro, R-568 dose-dependently blunted renin release, but also reduced the increase in renin due to forskolin (P < 0.01). Both isoproterenol and enalapril increased in vivo PRA to 3.1 +/- 0.3 and 3.7 +/- 0.5 ng Ang I/ml per h, respectively (P < 0.01), compared with vehicle (1.5 +/- 0.2 ng Ang I/ml per h). R-568 significantly reduced PRA to 2.1 +/- 0.1 ng/ml per h in isoproterenol-treated rats and to 1.6 +/- 0.2 ng/ml per h in enalapril-treated rats (P < 0.05). In low-salt treated animals, acute infusion of furosemide increased PRA from 8.7 +/- 3.2 to 18.6 +/- 2.3, whereas R-568 partially blunted this rise to 11.2 +/- 1.5 (P = 0.02). In vivo, R-568 significantly lowered serum calcium and PTH1-84, but the drug-induced changes in PRA were independent of the changes in calcium and parathyroid hormone. CONCLUSION: After the recent discovery of CaSRs in juxtaglomerular cells of mice, our results confirm the presence of such receptors in rats and demonstrate that these receptors modulate renin release both in vitro and in vivo. This suggests that CaSRs play a role as a regulatory pathway of renin release.

Haemodynamic consequences of changing bicarbonate and calcium concentrations in haemodialysis fluids.

BACKGROUND: In a previous study we demonstrated that mild metabolic alkalosis resulting from standard bicarbonate haemodialysis induces hypotension. In this study, we have further investigated the changes in systemic haemodynamics induced by bicarbonate and calcium, using non-invasive procedures. METHODS: In a randomized controlled trial with a single-blind, crossover design, we sequentially changed the dialysate bicarbonate and calcium concentrations (between 26 and 35 mmol/l for bicarbonate and either 1.25 or 1.50 mmol/l for calcium). Twenty-one patients were enrolled for a total of 756 dialysis sessions. Systemic haemodynamics was evaluated using pulse wave analysers. Bioimpedance and BNP were used to compare the fluid status pattern. RESULTS: The haemodynamic parameters and the pre-dialysis BNP using either a high calcium or bicarbonate concentration were as follows: systolic blood pressure (+5.6 and -4.7 mmHg; P < 0.05 for both), stroke volume (+12.3 and +5.2 ml; P < 0.05 and ns), peripheral resistances (-190 and -171 dyne s cm(-5); P < 0.05 for both), central augmentation index (+1.1% and -2.9%; ns and P < 0.05) and BNP (-5 and -170 ng/l; ns and P < 0.05). The need of staff intervention was similar in all modalities. CONCLUSIONS: Both high bicarbonate and calcium concentrations in the dialysate improve the haemodynamic pattern during dialysis. Bicarbonate reduces arterial stiffness and ameliorates the heart tolerance for volume overload in the interdialytic phase, whereas calcium directly increases stroke volume. The slight hypotensive effect of alkalaemia should motivate a probative reduction of bicarbonate concentration in dialysis fluid for haemodynamic reasons, only in the event of failure of classical tools to prevent intradialytic hypotension.

Influence of nutrition on urinary oxalate and calcium in preterm and term infants.

Few data for normal urinary oxalate (Ox) and calcium (Ca) excretion related both to gestational age and nutritional factors have been reported in preterm or term infants. We therefore determined the molar Ox and Ca to creatinine (Cr) ratios in spot urines from 64 preterm and 37 term infants aged 1-60 days, either fed formula or human milk (HM). Only vitamin D was supplemented; renal or metabolic diseases were excluded. Urinary Ox/Cr ratio was higher in preterm than in term infants, both when formula fed (1st month 253 vs. 180 mmol/mol and 2nd month 306 vs. 212 mmol/mol; P&lt;0.05) or HM fed (206 vs. 169 mmol/ mol and 283* vs. 232 mmol/mol; *P&lt;0.05). Ox/Cr was also higher in formula- than HM-fed preterm infants. The ratio increased during the first 2 months of life irrespective of nutrition. Urinary Ca/Cr ratio was comparable in all groups during the 1st month of life, except for a lower (P &lt; 0.05) value in term infants fed HM (0.10 mol/mol). It increased in all groups during the 2nd month of life, being highest in HM-fed preterm infants (1.86 mol/mol). In conclusion, urinary Ox and Ca excretion is influenced by both gestational age and nutrient intake in preterm and term infants.

High sensitivity of immature GABAergic neurons to blockers of voltage-gated calcium channels.

The involvement of voltage-gated calcium channels in the survival of immature CNS neurons was studied in aggregating brain cell cultures by examining cell type-specific effects of various channel blockers. Nifedipine (10 microM), a specific blocker of L-type calcium channels, caused a pronounced and irreversible decrease of glutamic acid decarboxylase activity, whereas the activity of choline acetyltransferase was significantly less affected. Flunarizine (1-10 microM, a relatively unspecific ion channel blocker) elicited similar effects, that were attenuated by NMDA. The glia-specific marker enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase, were affected only after treatment with high concentrations of nifedipine (50 microM) or NiCl2 (100 microM, shown to block T-type calcium channels). Nifedipine (50 microM), NiCl2 (100 microM), and flunarizine (5 microM) also caused a significant increase in the soluble nucleosome concentration, indicating increased apoptotic cell death. This effect was prevented by cycloheximide (1 microM). Furthermore, the combined treatment with calcicludine (10 nM, blocking L-type calcium channels) and funnel-web spider toxin-3.3 (100 nM, blocking T-type channels) also caused a significant increase in free nucleosomes as well as a decrease in glutamic acid decarboxylase activity. In contrast, cell viability was not affected by peptide blockers specific for N-, P-, and/or Q-type calcium channels. Highly differentiated cultures showed diminished susceptibility to nifedipine and flunarizine. The present data suggest that the survival of immature neurons, and particularly that of immature GABAergic neurons, requires the sustained entry of Ca2+ through voltage-gated calcium channels.

Phosphate forms in plant and their internal buffering in five soybean cultivars

Differences among plants in their ability to support nutritional stress periods may be caused by a differential vacuole capacity of ion storage and release and may also depend on the intensity of nutrient re-translocation under such conditions. In five soybean cultivars, submitted to eight days of P deprivation, the dry matter production and the contents of three phosphorus (P) forms - inorganic (Pi), organic (Po), and acid-soluble total (Pts) of different plant organs were determined. Pi release velocity (RSPi) was estimated as the tangent to the equations obtained for Pi f(t) at the point t = 2 days (the mean point in the period of greatest Pi decrease), considering that -deltaPi/deltat expresses the rate of Pi release. The internal Pi buffering capacity (IBCPi) was calculated as the inverse of the RSPi. Cultivars' differences in size of the non-metabolic Pi pool, RSPi, and the ability to transport Pi from less to more actively metabolizing regions were evaluated. The preferential Pi source and sink compartments under limited P absorption conditions were also evaluated. The cultivar Santa Rosa showed the highest Pi storage ability when the external supply was high, and a more intensive release under low P supply conditions than IAC8 and UFV1. The cultivar Uberaba was superior to Doko in its ability to store and use Pi. In all cultivars, upper leaves and roots were the main sink of Pi stored in the middle and lower leaves. Roots and upper leaves showed larger RSPi and lower IBCPi values than middle and lower leaves.

Interactions between magnesium, calcium, and aluminum on soybean root elongation

Alleviation of Al rhizotoxicity by Ca and Mg can differ among species and genotypes. Root elongation of soybean [Glycine max (L.) Merr.] line N93-S-179 and cvs. Young and Ransom exposed to varying concentrations of Al, Ca and Mg were compared in two experiments using a vertically split root system. Roots extending from a surface compartment with limed soil grew for 12 days into a subsurface compartment with nutrient solution treatments maintained at pH 4.6 with either 0 or 15 µmol L-1 Al. Calcium and Mg concentrations in treatments ranging from 0 to 20 mmol L-1. Although an adequate supply of Mg was provided in the surface soil compartment for soybean top growth, an inclusion of Mg was necessary in the subsurface solutions to promote root elongation in both the presence and absence of Al. In the absence of Al in the subsurface solution, tap root length increased by 74 % and lateral root length tripled when Mg in the solutions was increased from 0 to either 2 or 10 mmol L-1. In the presence of 15 µmol L-1 Al, additions of 2 or 10 mmol L-1 Mg increased tap root length fourfold and lateral root length by a factor of 65. This high efficacy of Mg may have masked differences in Al tolerance between genotypes N93 and Young. Magnesium was more effective than Ca in alleviating Al rhizotoxicity, and its ameliorative properties could not be accounted for by estimated electrostatic changes in root membrane potential and Al3+ activity at the root surface. The physiological mechanisms of Mg alleviation of Al injury in roots, however, are not known.

Methamphetamine Inhibitor: Calcium Nitrate– What You Need to Know, 2006

Iowa agriculture depends on anhydrous ammonia as a low-cost form of nitrogen fertilizer on 61 percent of Iowa’s 12.4 million acres of corn. Now we find a threat to that source of nutrient—the theft of anhydrous ammonia for use in making a powerful, illegal narcotic called methamphetamine. Naturally, the fertilizer industry is outraged by the illegal and illicit use of our products. We want to play a role in preventing abuse in the future. By raising awareness, knowing how to respond and using the Meth Inhibitor, fertilizer dealers can assist law enforcement in combating this illicit use of a product important to Iowa farmers.

A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.

PDMP blocks brefeldin a-induced retrograde membrane transport from Golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not ¿rescue¿ the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

A link between eumelanism and calcium physiology in the barn owl.

In many animals, melanin-based coloration is strongly heritable and is largely insensitive to the environment and body condition. According to the handicap principle, such a trait may not reveal individual quality because the production of different melanin-based colorations often entails similar costs. However, a recent study showed that the production of eumelanin pigments requires relatively large amounts of calcium, potentially implying that melanin-based coloration is associated with physiological processes requiring calcium. If this is the case, eumelanism may be traded-off against other metabolic processes that require the same elements. We used a correlative approach to examine, for the first time, this proposition in the barn owl, a species in which individuals vary in the amount, size, and blackness of eumelanic spots. For this purpose, we measured calcium concentration in the left humerus of 85 dead owls. Results showed that the humeri of heavily spotted individuals had a higher concentration of calcium. This suggests either that plumage spottiness signals the ability to absorb calcium from the diet for both eumelanin production and storage in bones, or that lightly spotted individuals use more calcium for metabolic processes at the expense of calcium storage in bones. Our study supports the idea that eumelanin-based coloration is associated with a number of physiological processes requiring calcium.

Calcium homeostasis in the central nervous system: adaptation to neurodegeneration.

Here we review the results of our recent studies on neurodegeneration together with data on cerebral calcium precipitation in animal models and humans. A model that integrates the diversity of mechanisms involved in neurodegeneration is presented and discussed based on the functional relevance of calcium precipitation.