William Pralong - Academia.edu (original) (raw)

Papers by William Pralong

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

Journal of Biological Chemistry, Feb 1, 1991

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F ... more The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.

Diabetes, Jul 1, 1998

A n i m a l s. Male Wistar rats, age 3 months and weighing 220-240 g, were used. They had free ac... more A n i m a l s. Male Wistar rats, age 3 months and weighing 220-240 g, were used. They had free access to water and standard laboratory diet pellets (No. 113; Usine d'Alimentation Rationnelle, Vi l l e m o i s s o n-s u r-Orge, France). Induction of experimental diabetes. Diabetes was induced by a single intravenous dose of 35 mg/kg body wt STZ (Sigma, St. Louis, MO) dissolved in a citrate buffer (0.1 mol/1; pH 4.5) that was injected through the saphenous vein (8). Controls were intravenously injected with citrate buffer. The diabetic state was characterized by the measurement of basal plasma glucose concentration and an intravenous glucose tolerance test (IVGTT) performed 2 weeks after STZ injec-From the

Journal of Neurochemistry, May 8, 2003

Analytical Biochemistry, Aug 1, 1998

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat... more We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [ 3 H]glycogen from D-[6-3 H]glucose. The dose-response curve of insulin in this assay exhibited an EC 50 of 0.42 (؎0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A 1-13 B 1-9 , A 1-14 B 1-9 , and A 14-21 B 14-30 showed no measurable activity. Although preparations of fragment A 14-21 B 10-30 displayed dose-dependent agonist activity with an EC 50 of 380 (؎40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.

Journal of Biological Chemistry, Mar 1, 1992

Journal of Neuroendocrinology, Jun 1, 1991

Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, a... more Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration, [Ca"],. With an appropriate selection of the excitation wavelengths, in dual excitation rnicrofluorimetry experiments, it was possible to distinguish between fluorescence changes due to altered [Ca"], versus quinacrine exocytosis. Transient elevations of [Ca"], were provoked in individual pituitary cells by enhancing calcium influx through voltage gated channels. In part of the cells an initial increase in [Ca"], coincided with stimulated quinacrine release. The approach was also applied to cells of the neuroblastoma line NCB20, where stimulation with bradykinin caused a transient rise in [Ca" I,, concomitantly with enhanced exocytosis. No increase in exocytosis was ever detected without an elevation of [Ca"],, suggesting that in both cellular systems, an increase in [Ca"], is absolutely necessary, but not sufficient to induce secretion.

Biomaterials, Aug 1, 2004

The encapsulation of genetically modified cells represents a promising approach for the delivery ... more The encapsulation of genetically modified cells represents a promising approach for the delivery of therapeutic proteins. The functionality of the device is dependent on the characteristics of the biomaterials, the procedures used in its confection and the adaptability of the encapsulated cells in the host. We report conditions leading to the development of calcifications on the polyvinyl alcohol (PVA) matrix introduced in hollow fiber devices for the encapsulation of primary human fibroblasts implanted in mice. The manufacturing procedures, batches of PVA matrix and cell lineages were assessed for their respective role in the development of the phenomenon. The results showed that the calcification is totally prevented by substituting phosphate-buffer saline with ultra-pure sterile water in the rinsing procedure of the matrix. Moreover, a positive correlation was found, when comparing two fibroblast cell lineages, between the level of lactate dehydrogenase (LDH) activity measured in the cells and the degree of calcium deposition. Higher LDH activity may decrease calcium depositions because it generates in the device a more acidic microenvironment inhibiting calcium precipitation. The present study defines optimized conditions for the encapsulation of primary human fibroblasts in order to avoid potentially detrimental calcifications and to allow long-term survival of encapsulated cells.

Cytoplasmic calcium ions and other signalling events in insulin secretion

Biochemical Society Transactions, Feb 1, 1990

Survival of Encapsulated Human Primary Fibroblasts and Erythropoietin Expression Under Xenogeneic Conditions

Human Gene Therapy, Jul 1, 2004

Allogeneic cells are the most attractive source for cell transplantation, as the use of xenogenei... more Allogeneic cells are the most attractive source for cell transplantation, as the use of xenogeneic cells is hampered by safety concerns and the use of autologous cells involves practical difficulties. The immune rejection of allogeneic cells can be overcome by physical immunoprotection provided by polymer encapsulation. To study the variability of cell and donor sources, we compared different primary human cells as candidates for gene therapy-mediated delivery of human erythropoietin (hEpo). DARC-3.1 fibroblasts, MDX-01 fibroblasts, and ARPE-19 retinal pigment epithelial cells were encapsulated into polyethersulfone hollow fibers and implanted for 1 month in nude mice as well as in immunocompetent and FK506-immunosuppressed mice to test their in vivo resistance, with the assumption that xenogeneic conditions constitute a stringent model for human application. DARC-3.1 fibroblasts showed the best survival, prompting us to evaluate cell lineages from the same donor (DARC-3.2) or another donor (DARC-4.3 and DARC-4.4). With the exception of DARC-4.3, the remaining three lineages showed comparable survival in immunocompetent C3H and DBA/2J mice. DARC-3.1 fibroblasts were retrovirally engineered with hEpo cDNA, reaching a secretion level of 170 IU of hEpo per 10(6) cells per day. Encapsulated DARC-3.1-hEpo cells led to significantly increased hematocrits in the various hosts and under various transplantation conditions. The present study shows that encapsulated primary human DARC-3.1 fibroblasts are able to survive under xenogeneic conditions and, once engineered with hEpo cDNA, to increase the hematocrit of transplanted mice.

Endocrinology, May 1, 1996

Signal transduction of two mitogens for pancreatic p-cells, GH and PRL, was investigated using th... more Signal transduction of two mitogens for pancreatic p-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-l. Addition of human GH (hGH) or ovine PRL in a serumsubstitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated ["Hlthymidine incorporation in a dose-dependent manner (1 PM-1 nM). hGH induced cytosolic Ca' ' (ICa" I,) rises, which were transient, dependent on the presence of extracellular Ca' +, blocked by verapamil, calciseptinc, and the hvperpolarizing agent diazoxide, SUEgesting that h&H stimulates Ca" '-&flux thr&gi L-type Caz'-thannels. Similar effects on ICaZ' I. were observed with bGH or PRL. hGH caused membrane depblarization in a small proportion of the cells (~25%) as detected by cell-attached patch-clamp analysis. However,

Activity-Dependent Phosphorylation of SNAP-25 in Hippocampal Organotypic Cultures

Journal of Neurochemistry, Dec 25, 2001

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exoc... more Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

Pyridine nucleotide redox state parallels production of aldosterone in potassium-stimulated adrenal glomerulosa cells

Proceedings of the National Academy of Sciences of the United States of America, 1992

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP

Journal of Biological Chemistry, Sep 1, 1992

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancre... more In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.

Dynamic pacing of cell metabolism by intracellular Ca2+ transients

Journal of Biological Chemistry, Nov 1, 1994

During cell activation, Ca2+, by stimulating the NADH-producing mitochondrial dehydrogenases, tri... more During cell activation, Ca2+, by stimulating the NADH-producing mitochondrial dehydrogenases, triggers the generation of reducing equivalents whereby ATP production is sustained. In cell populations, [Ca2+] changes in the mitochondrial matrix were demonstrated to parallel rapidly those in the cytosol ([Ca2+]i). There is still no indication as to whether metabolic activation follows oscillatory patterns similar to those of [Ca2+]i. Therefore, changes in NAD(P)H were monitored in single pancreatic beta-cells, adrenal glomerulosa cells, and liver cells during oscillatory [Ca2+]i transients. Rapid NAD(P)H and [Ca2+]i oscillations with similar frequency and sensitive both to changes in glucose concentration and to extracellular Ca2+ removal were identified in a subpopulation of pancreatic beta-cells in primary culture. Furthermore, Ca(2+)-dependent oscillatory NAD(P)H formation could be evoked by the pulsatile application of depolarizing [K+], demonstrating the pacing effect of increased [Ca2+]i on beta-cell metabolism. In adrenal glomerulosa cells, angiotensin II, a physiological stimulator of aldosterone production, could be shown to elicit the oscillatory formation of mitochondrial NAD(P)H through frequency modulation of [Ca2+]i transients. In contrast to the two former endocrine cell types, in hepatocytes, [Arg8]vasopressin and epinephrine caused the amplitude modulation of NAD(P)H formation. Taken together, these results provide unprecedented evidence for a cell-specific pacing of metabolism by [Ca2+]i transients coordinated with cell activation and function.

The Journal of Membrane Biology, Jun 1, 1986

A new technique for continuous monitoring of the cellular calcium was developed and used for stud... more A new technique for continuous monitoring of the cellular calcium was developed and used for studying the effects of external and internal Na (Nao and Nai), external Ca (Cao), Ca ionophore A23187, and electrical activity on membrane-bound and intracellular Ca in mammalian nonmyelinated nerve fibers. Increasing Cao increased both the membrane-bound and the intracellular Ca. Lowering Nao increased the membrane-bound fraction of Ca indicating that lack of Na, enhanced the capacity of the plasma membrane to bind Ca, and produced an increase of the internal Ca pool. Increasing Nai by treatment with ouabain enhanced the Ca inflow in both, the presence and absence of Nao, presumably by stimulating the Cao/Nai exchange. The Ca ionophore A23187 produced a large and irreversible increase in the intracellular Ca without affecting the membrane-bound fraction. On the other hand, electrical activity, which is known to produce a large increase of the total Ca in squid axon, had no measurable effect on the total calcium content in our preparation. It is concluded that in mammalian nerve fibers a Ca load by exposition to Na-free solution or to A23187 produces an accumulation of Ca into the intracellular Ca stores, whereas during electrical activity the membrane-associated extrusion mechanisms are able to maintain the intracellular Ca 2+ below the threshold for intracellular sequestration. Furthermore, the results indicate that the intracellular sequestration mechanisms are dependent on the internal concentration of Na.

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

The Journal of Membrane Biology, Jul 1, 1988

Calcium efflux was measured in desheathed rabbit vagus nerves loaded with 45Ca2+. The effects of ... more Calcium efflux was measured in desheathed rabbit vagus nerves loaded with 45Ca2+. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of 45Ca2+ efflux profiles. The 45Ca2+ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min-1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min-1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm-2 sec-1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.

Apoptosis, 1997

RIN cells were infected with recombinant Semliki Forest virus (SFV) particles containing the LacZ... more RIN cells were infected with recombinant Semliki Forest virus (SFV) particles containing the LacZ gene. X-gal staining showed 100% infectivity of the cell cultures and high-level expression of bacterial β-galactosidase in these cells. The cytopathogenic effects of the SFV infection were studied by measuring the viability of the RIN cells. Comparisons between control RIN cells and Bcl-2 overexpressing RIN cells were done 72 h post-infection. Significant differences in viability levels were observed. The control RIN cells showed in the MTT assay a mean value of 0.156±0.017 compared to 0.347±0.057 for the RIN/Bcl-2 cells. FACS analysis of cells labelled with propidium iodide indicated that only an average of 4.5±0.5% of the control cells were viable 72 h post-infection, while 44.5±3.5% of the RIN/Bcl-2 cells were still alive. Thus, the Bcl-2 overexpression clearly protected the SFV-infected cells from undergoing apoptosis.

FEBS Letters, Mar 12, 1999

Caffeine mobilized an intracellular Ca P+ pool in intact fura-2-loaded INS-1 cells in suspension ... more Caffeine mobilized an intracellular Ca P+ pool in intact fura-2-loaded INS-1 cells in suspension exposed to high (16 mM) [glucose], while a minor effect was observed with low (2 mM) [glucose]. Cells were kept in a medium containing diaxozide or no Ca P+ to prevent the influx of extracellular Ca P+. The caffeine-sensitive intracellular Ca P+ pool was within the endoplasmic reticulum since it was depleted by the inhibitor of the reticular Ca P+ pumps thapsigargin and the InsP Q-dependent agonist carbachol. No effect of caffeine was observed in the parent glucose-insensitive RINmF5 cells. In microsomes from INS-1 but not RINmF5 cells, the type 2 ryanodine receptor was present as revealed by Western blotting. It was concluded that the endoplasmic reticulum of INS-1 cells possesses caffeinesensitive type 2 ryanodine receptors Ca P+ channels.

Experimental Neurology, May 1, 2005

Adenosine is an important inhibitory modulator of brain activity. In a previous ex vivo gene ther... more Adenosine is an important inhibitory modulator of brain activity. In a previous ex vivo gene therapy approach, local release of adenosine by encapsulated fibroblasts implanted into the vicinity of an epileptic focus, was sufficient to provide transient protection from seizures (

The EMBO Journal, Nov 1, 1994

The cx-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an imp... more The cx-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting ,3 cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of , cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.