Juan A. Reig

Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells

Embryonic stem cells possess the ability to differentiate in vitro into a variety of cell lineages, including insulin‐producing cells. Pancreatic β‐cells derive from foregut endoderm during embryonic development. However, previous reports using transgenic mice strongly indicate that insulin‐positive cells may be generated also through the neuroectoderm pathway. To analyze this point, a culture system was performed in which only ectoderm committed cells were present. Based on published work, we achieved this by maintaining transfected clonal R1 mouse embryonic stem cells in monolayer in the absence of LIF. Contrary to differentiation protocols via embryoid body formation, monolayer cultured cells displayed ectodermal fates according to the marker gene expression pattern. Under these particular conditions, neomycin was added in order to select insulin‐expressing cells. The cell lineage obtained expressed Pdx1, Pax6, Isl1, AChE, MBP, TH, and GS genes, confirming ectodermal commitment, even though some of these factors are also expressed in endoderm. In addition these cells displayed excitatory properties similar to astrocytes. Co‐expression of insulin II and nestin was observed in monolayer culture and in the presence of specific conditioned media. No expression of early endodermal markers was detected along monolayer cultures. Altogether, these observations suggest that cells with ectoderm fates could participate in vitro in the derivation of insulin‐producing cells. These results have implications for insulin gene regulation and hormone secretion in order to generate insulin‐producing cells for replacement protocols in the treatment of diabetes.

A peptide that mimics the carboxy‐terminal domain of SNAP‐25 blocks Ca2+‐dependent exocytosis in chromaffin cells

SNAP‐25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle‐plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP‐25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti‐SNAP‐25 monoclonal antibody to show that SNAP‐25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca2+‐evoked catecholamine release from digitonin‐permeabilized chromaffin cells in a time‐ and dose‐dependent manner. Remarkably, a 20‐mer synthetic peptide representing the sequence of the C‐terminal domain of SNAP‐25 blocked Ca2+‐dependent catecholamine release with an IC50 = 20 μM. The inhibitory activity of the peptide was sequence‐specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C‐terminal segment of SNAP‐25, therefore, plays a key role in regulating Ca2+‐dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.

Separate Binding and Functional Sites for ω co‐Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Abstract: Purified adrenomedullary plasma membranes contain two high‐affinity binding sites for l25I‐ω‐conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin‐receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled ω‐conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two‐site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cho‐linoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration‐dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmaxof 129 fmol/mg of protein. At 0.25 μM, co‐conotoxin was notable to block depolarization‐evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mMK+for 30 s, whereas under the same conditions, 1 μM nitrendipine inhibited uptake by ∼60%. When cells were hyper‐polarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mMK+, ω‐conotoxin partially inhibited Ca2+ uptake in a concentration‐dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of ω‐conotoxin binding sites remains to be determined.

Role of syntaxin in mouse pancreatic beta cells

SummaryThe role of syntaxin 1, a protein involved in the docking of synaptic vesicles at presynaptic active zones, has been investigated in pancreatic islet cells. Using two different monoclonal antibodies we have shown that syntaxin 1 is present in the pancreatic islet cell microsomal fraction. Furthermore, functional experiments demonstrate that anti-syntaxin antibodies inhibit Ca2+-dependent insulin secretion in permeabilized islet cells. These data indicate that syntaxin 1 is present in the pancreatic beta cell and it is likely to play a functional role in the exocytosis of secretory granules.

ISOLATION AND CHARACTERIZATION OF RESIDUAL UNDIFFERENTIATED MOUSE EMBRYONIC STEM CELLS FROM EMBRYOID BODY CULTURES BY FLUORESCENCE TRACKING

SummaryThe differentiation of mouse embryonic stem (ES) cells can be induced in vitro after leukemia inhibitory factor (LIF) withdrawal and further enhanced by the formation of “embryoid body” (EB) aggregates. This strategy is being used in order to optimize differentiation protocols that would result in functional cells for experimental cell replacement therapies. However, this study presents the possibility for residual undifferentiated cells to survive after standard in vitro procedures. Mouse ES cells were stably transfected with the enhanced green fluorescent protein (EGFP), under the control of the Oct4 promoter, a transcription factor that is expressed in undifferentiated ES cells but down-regulated on differentiation. Residual fluorescent cells were isolated from EBs that were cultured in standard conditions in absence of LIF. These residual cells displayed recurrent gain of chromosomes 8 and 9. Residual fluorescent cells, further expanded in absence of LIF and cultured as EBs, still displayed a significant Oct4 expression in comparison with parental transfected ES cells. Consequently, these residual cells have an intrinsic resistance to differentiate. The behavior of these cells, observed in vitro, can be overcome in vivo, as they were able to induce teratomas in subcutaneously injected nude mice. Residual undifferentiated cells displayed slight levels of VASA and DAZL expression. These results demonstrate that mouse ES cells cultured in vitro, in standard conditions, can spontaneously acquire recurrent karyotypical changes that may promote an undifferentiated stage, being selected in standard culture conditions in vitro.

Ruthenium red inhibits selectively chromaffin cell calcium channels

The effect of Ruthenium red (RR) on ionic currents and catecholamine secretion was studied in chromaffin cells. This polycation inhibited 59 mM potassium-stimulated 45Ca2+ uptake in a concentration-dependent manner (IC50 = 5 +/- 0.2 microM). This effect was more evident at extracellular calcium concentrations over 1 mM and was not abolished by neuraminidase pretreatment. RR also inhibited potassium-stimulated catecholamine secretion (IC50 = 6 +/- 0.9 microM). These results were corroborated by patch-clamp in whole-cell recordings. RR inhibited chromaffin cell calcium currents (IC50 = 7 microM) without affecting significantly either sodium or potassium currents. Radioligand binding studies in adrenomedullary plasma membranes showed that RR inhibited [125I]omega-conotoxin GVIA binding but it had no effect on specific binding of [3H]nitrendipine. The effect of the RR on calcium currents was additive with the inhibitory effect observed with 10 microM nitrendipine. The residual dihydropyridine-resistant calcium current was inhibited with a potency similar to that determined under control conditions in the absence of nitrendipine. These results demonstrate that RR selectively inhibits calcium channels; however, this polycation was not selective for a particular calcium channel subtype.

Nutrient toxicity in pancreatic β-cell dysfunction.

Nutrients, such as glucose and fatty acids, have a dual effect on pancreatic β-cell function. Acute administration of high glucose concentrations to pancreatic β-cells stimulates insulin secretion. In addition, short term exposure of this cell type to dietary fatty acids potentiates glucose-induced insulin release. On the other hand, long-term exposure to these nutrients causes impaired insulin secretion, characterized by elevated exocytosis at low concentrations of glucose and no response when glucose increases in the extracellular medium. In addition, other phenotypic changes are observed in these conditions. One major step in linking these phenotypic changes to the diabetic pathology has been the recognition of both glucose and fatty acids as key modulators of β-cell gene expression. This could explain the adaptative response of the cell to sustained nutrient concentration. Once this phase is exhausted, the β-cell becomes progressively unresponsive to glucose and this alteration is accompanied by the irreversible induction of apoptotic programs. The aim of this review is to present actual data concerning the development of the toxicity to the main nutrients glucose and fatty acids in the pancreatic β-cell and to find a possible link to the development of type 2 diabetes.ResumenTanto la glucosa como los ácidos grasos tienen un doble efecto sobre la función de la célula β pancreática. La administración aguda de elevadas concentraciones de glucosa a la célula β estimula la secreción de insulina. Además, una corta exposición de esta célula a los ácidos grasos de la dieta potencia la liberación de insulina inducida por glucosa. Por otra parte, la exposición prolongada a estos nutrientes causa alteraciones en la secreción de insulina, caracterizada por una elevada exocitosis a bajas concentraciones de glucosa y una falta de respuesta cuando la glucosa incrementa en el medio extracelular. Además, otros cambios fenotípicos aparecen en estas circunstancias. Un paso importante a la hora de relacionar estos cambios fenotípicos con la patología diabética ha sido la identificación de la glucosa y los ácidos grasos como controladores esenciales en la expresión génica en la célula β. Esto podría explicar la respuesta adaptativa de la célula a las elevadas concentraciones de nutrientes. Una vez esta fase se ha agotado, la célula β se vuelve progresivamente insensible a la glucosa y esta alteración viene acompañada por una inducción irreversible de programas apoptóticos. El objetivo de esta revisión es presentar datos actuales concernientes al desarrollo de la toxicidad a los principales nutrientes glucosa y ácidos grasos en la célula β pancreática y encontrar una posible relación con el desarrollo de la diabetes tipo 2.

A Two-Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus

Abstract: Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two‐dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two‐dimensional electrophoretograms were studied with the aid of computer‐assisted image analysis (CAIA). A database map of 32P‐labeled proteins was constructed; ∼500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and iso‐electric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+‐dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC 12 ceils.

Naphthalenesulfonamide derivatives ML9 and W7 inhibit catecholamine secretion in intact and permeabilized chromaffin cells

The role of protein phosphorylation in catecholamine secretion from bovine adrenomedullary chromaffin cells was studied using different protein kinase inhibitors. Naphthalenesulfonamide derivatives as ML9 and ML7, more specific for the myosin light chain kinase, and the calmodulin antagonist W7 inhibited catecholamine secretion 20 and 40% respectively in digitonin-permeabilized chromaffin cells. ML9 also decreased calcium evoked protein phosphorylation of different proteins including tyrosine hydroxylase in permeabilized cells. These naphthalenesulfonamide derivatives showed also an effect in intact cells, ML9 and W7 produced 50% inhibition in catecholamine secretion and45Ca2+ uptake, however H8 had no effect. The partial [3H]nitrendipine binding displacement of these drugs to adrenomedullary membranes suggests that these sulfonamide derivatives could interact directly with L-type calcium channels in intact cells. The results obtained in permeabilized cells suggest a possible role of protein phosphorylation in the regulation of catecholamine secretion in chromaffin cells.

Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla

1 Two binding sites for the 1,4‐dihydropyridine (DHP) derivative [3H]‐nitrendipine have been found in the bovine adrenal medulla. The high‐affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg−1 protein) was specifically located in purified plasma membranes. The low‐affinity site (Kd = 252nM and Bmax = 169 pmol mg−1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]‐nitrendipine. 2 Kinetic analysis of the rates of association and dissociation of [3H]‐nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200‐110 revealed single, homogeneous populations of high‐ and low‐affinity sites in plasma and mitochondrial membranes, respectively. 3 The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]‐nitrendipine. 4 In contrast, binding of [3H]‐nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]‐nitrendipine acting in a non‐competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]‐nitrendipine. 5 These results suggest that both binding sites reflect different receptor entities. The high‐affinity binding site corresponds to the dihydropyridine receptor associated with the L‐type calcium channel. The function of the mitochondrial, low‐affinity binding site is, at present, unknown.