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P Meda

Publications and source records attributed to P Meda.

At least 37 records · Page 2Linked to original sources
PubMed2000

Differential expression of connexins during stratification of human keratinocytes.

To assess whether gap junctions and connexins change during keratinocyte differentiation, we have studied epidermal equivalents obtained in organotypic cultures of keratinocytes from the outer root sheath of human hair follicles. These reconstituted tissues exhibit a number of differentiation and proliferation markers of human epidermis, including gap junctions, connexins, and K6 and Ki67 proteins. Immunostaining and northern blots showed that gap junctions of the epidermal equivalents were made of Cx26 and Cx43. Cx26 was expressed in all keratinocyte layers, throughout the development of the epidermal equivalents. In contrast, Cx43 was initially observed only in the basal layer of keratinocytes and became detectable in the stratum spinosum and granulosum only after the epidermal equivalents had thickened. The levels of Cx26 and its transcript markedly increased as a function of stratification of the epidermal equivalents, whereas those of Cx43 remained almost constant. Microinjection of Lucifer Yellow into individual keratinocytes showed that gap junctions were similarly permeable at all stages of development of the epidermal equivalents. The data show that epidermal equivalents (i) feature a pattern of connexins typical of an actively renewing human interfollicular epidermis, and (ii) provide a model that reproduces the tridimensional organization of intact epidermis and that is amenable for experimentally testing the function of junctional communication between human keratinocytes.

Cell Differentiation↗
PubMed2000

Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance.

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.

Animals↗
PubMed2000

Glucose regulates proinsulin and prosomatostatin but not proglucagon messenger ribonucleic acid levels in rat pancreatic islets.

Insulin and glucagon are the major hormones involved in the control of fuel metabolism and particularly of glucose homeostasis; in turn, nutrients tightly regulate insulin and glucagon secretion from the islets of Langerhans. Nutrients have clearly been shown to affect insulin secretion, as well as insulin biosynthesis and proinsulin gene expression; by contrast, the effects of nutrients on proglucagon gene expression have not been studied. We have investigated the effect of glucose, arginine, and palmitate on glucagon release, glucagon cell content, and proglucagon messenger RNA (mRNA) levels from isolated rat islets in 24-h incubations. We report here that concentrations of glucose that clearly regulate insulin and somatostatin release as well as proinsulin and prosomatostatin mRNA levels, do not significantly affect glucagon release, glucagon cell content or proglucagon mRNA levels. In addition, though both 10 mM arginine and 1 mM palmitate strongly stimulated glucagon release, they did not affect proglucagon mRNA levels. We conclude that, in contrast to insulin and somatostatin, glucose does not affect glucagon release and proglucagon mRNA levels, and arginine and palmitate do not coordinately regulate glucagon release and proglucagon mRNA levels.

Animals↗
PubMed2000

Chronic hypertension alters the expression of Cx43 in cardiovascular muscle cells.

Connexin43 (Cx43), the predominant gap junction protein of muscle cells in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension on the expression of Cx43 in aorta and heart in three different models of experimental hypertension. Rats were made hypertensive either by clipping one renal artery (two kidney, one-clip renal (2K,1C) model) by administration of deoxycorticosterone and salt (DOCA-salt model) or by inhibiting nitric oxide synthase with N G-nitro-L-arginine methyl ester (L-NAME model). After 4 weeks, rats of the three models showed a similar increase in intra-arterial mean blood pressure and in the thickness of the walls of both aorta and heart. Analysis of heart mRNA demonstrated no change in Cx43 expression in the three models compared to their respective controls. The same 2K,1C and DOCA-salt hypertensive animals expressed twice more Cx43 in aorta, and the 2K, 1C rats showed an increase in arterial distensibility. In contrast, the aortae of L-NAME hypertensive rats were characterized by a 50% decrease in Cx43 and the carotid arteries did not show increased distensibility. Western blot analysis indicated that Cx43 was more phosphorylated in the aortae of 2K,1C rats than in those of L-NAME or control rats, indicating a differential regulation of aortic Cx43 in different models of hypertension. The data suggest that localized mechanical forces induced by hypertension affect Cx43 expression and that the cell-to-cell communication mediated by Cx43 channels may contribute to regulating the elasticity of the vascular wall.

Animals↗
PubMed2000

Importance of cell-matrix interactions in rat islet beta-cell secretion in vitro: role of alpha6beta1 integrin.

It has long been recognized that islet cell function is rapidly altered in vitro, but can be maintained, at least in part, when cells are layered on defined extracellular matrices. The present work addresses the influence of short-term cell-matrix interactions on islet beta-cell function and provides first insight into the molecular basis of these interactions. When primary rat beta-cells were allowed to attach to a matrix produced by a rat carcinoma cell line (804G), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting beta-cells and in the amount of insulin secreted per active cell, as shown using the reverse hemolytic plaque assay. In turn, the spreading or flattening of beta-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. Using indirect immunofluorescence, it was found that 1)alpha6beta1 integrins are present at the surface of islet cells in situ, 2) alpha6beta1 expression is heterogeneous among purified beta-cells and is upregulated by insulin secretagogues, 3) alpha6beta1 expression is higher in spreading cells, and 4) anti-alpha6beta1-specific antibodies decrease spreading. These observations demonstrate that islet cell-matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by alpha6beta1 integrins, suggesting that outside-in signaling through alpha6beta1 integrin plays a major role in the regulation of beta-cell function.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Cx36 preferentially connects beta-cells within pancreatic islets.

Previous studies have provided evidence for the transcripts of Cx43 and Cx45 within pancreatic islets. As of yet, however, it has proven difficult to unambiguously demonstrate the expression of these proteins by islet cells. We have investigated whether Cx36, a new connexin species recently identified in mammalian brain and retina, may also be expressed in pancreatic islets. Using probes that permitted the original identification of Cx36 in the central nervous system, we show that a transcript for Cx36 is clearly detectable in rat pancreatic islets. Using novel and affinity-purified polyclonal antibodies, we have found that Cx36 is actually expressed in pancreatic islets. Both in situ hybridization and immunolabeling indicated that this connexin is abundant in the centrally located insulin-producing beta-cells and is expressed much less, if at all, by the other endocrine cell types. This differential expression was further confirmed on fluorescence-activated cell sorter-purified preparations enriched in either beta- or non-beta-cells. The finding of a differential distribution of Cx36 within distinct regions of pancreatic islets creates the possibility that this connexin may provide the establishment of selective pathways of communication between the different types of endocrine cells comprising the pancreatic islet.

Animals↗
PubMed1999

Modulation of pancreatic acinar cell to cell coupling during ACh-evoked changes in cytosolic Ca2+.

The temporal changes in cytosolic free Ca2+ ([Ca2+]i), Ca2+-dependent membrane currents (Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were measured in rat pancreatic acinar cells using digital imaging and dual perforated patch-clamp recording. ACh (50 nM-5 microM) increased [Ca2+]i and evoked Im currents without altering Ij in 19 of 37 acinar cell pairs. Although [Ca2+]i rose asynchronously in cells comprising a cluster, the delay of the [Ca2+]i responses decreased with increasing ACh concentrations. Perfusion of inositol 1,4,5-trisphosphate (IP3) into one cell of a cluster resulted in [Ca2+]i responses in neighboring cells that were not necessarily in direct contact with the stimulated one. This suggests that extensive coupling between acinar cells provides a pathway for cell-to-cell diffusion of Ca2+-releasing signals. Strikingly, maximal (1-5 microM) ACh concentrations reduced Ij by 69 +/- 15% (n = 9) in 25% of the cell pairs subjected to dual patch-clamping. This decrease occurred shortly after the Im peak and was prevented by incubating acinar cells in a Ca2+-free medium, suggesting that uncoupling was subsequent to the initiation of the Ca2+-mobilizing responses. Depletion of Ca2+-sequestering stores by thapsigargin resulted in a reduction of intercellular communication similar to that observed with ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with L-nitro-arginine and restored by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are regulated concurrently. Closure of gap junction channels may occur to functionally isolate nearby cells differing in their intrinsic sensitivity to ACh and thereby to allow for sustained activity of groups of secreting cells.

Acetylcholine↗
PubMed1999

Loss of alpha 1 connexin does not alter the prenatal differentiation of pancreatic beta cells and leads to the identification of another islet cell connexin.

Connexin alpha 1, also referred to as Cx43, has thus far been the only gap junction protein identified between the hormone-producing cells of pancreatic islets. To investigate whether loss of this connexin affects the development of endocrine pancreas and the differentiation of insulin-producing beta cells, we have taken advantage of a transgenic line in which the gene coding for connexin alpha 1 had been functionally deleted by homologous recombination. Analysis of pancreas at embryonal day 19.5 (E 19.5) after immunostaining for the four main types of islet hormones, showed that islet cell development was similar in homozygous transgenic mice that completely lacked alpha 1 connexin, in mice that were heterozygous for the transgene, and in age-matched controls with a genetic background similar to that of the transgenic animals. In particular, the three animal groups featured beta cells that had a similar insulin content and ultrastructural organization, including the presence of typical gap junction plaques on the membrane. However, quantitative analysis of freeze-fractured membranes showed that these plaques were less frequent in the transgenic mice lacking alpha 1 connexin. This finding prompted us to revisit the connexin pattern of normal pancreatic beta cells. Using RT-PCR amplification and primers specific for nine of the mammalian connexins, we have found that normal rat and mouse pancreas contain six connexin transcripts, including one that codes for alpha 6 connexin, a protein also referred to as Cx45. This transcript was also identified in isolated pancreatic islets, in FACS-purified suspensions of primary beta cells and in the insulin-producing cells of an experimental tumor. Using antibodies, we found that connexin alpha 6 is expressed by the latter cells, as well as by pancreatic fibroblasts and epithelial duct cells. The data show that pancreatic islets have a normal prenatal development in mice that no longer express alpha 1 connexin. They further provide evidence that normal and tumoral insulin-producing cells natively coexpress connexins alpha 1 and alpha 6.

Animals↗
PubMed1999

Aortic connexin43 is decreased during hypertension induced by inhibition of nitric oxide synthase.

Connexin43 (Cx43), the predominant gap junction protein in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension induced by inhibition of nitric oxide synthase on the expression of Cx43 in aorta and heart as well as on the distensibility of the carotid artery. Administration of 0.4 g/L NG-nitro-L-arginine methyl ester (L-NAME) to rats in their drinking water for 4 weeks increased intra-arterial mean blood pressure, wall thickness of aorta and carotid artery (25%), and heart weight (17%). Analysis of heart mRNA demonstrated increased expression of the fetal skeletal alpha-actin and of atrial natriuretic peptide but not of Cx43. In contrast, Cx43 mRNA and protein were decreased by 50% in the aortas of L-NAME-treated rats that did not show increased carotid distensibility. Because these data contrasted with those obtained in the 2-kidney, 1 clip model of rat hypertension, which is characterized by increased arterial distensibility and Cx43 expression in aorta, we investigated by Western blot analysis the posttranslational modifications of Cx43. We found that Cx43 was more phosphorylated in the aorta of 2-kidney, 1 clip rats than in that of L-NAME or control rats, which indicated a differential regulation of Cx43 in different models of hypertension. The data suggest that the cell-to-cell communication mediated by Cx43 channels may help regulate the elasticity of the vascular wall.

Animals↗
PubMed1999

Glucose-induced preproinsulin gene expression is inhibited by the free fatty acid palmitate.

Prolonged exposure to elevated FFA levels has been shown to induce peripheral insulin resistance and to alter the beta-cell secretory response to glucose. To investigate the effects of FFAs on preproinsulin gene expression, we measured insulin release, cell content, and messenger RNA (mRNA) levels in rat islets after a 24-h exposure to 1 mM palmitate. Insulin release increased at all glucose concentrations studied; in contrast, preproinsulin mRNA levels were specifically reduced by palmitate at high glucose with a decrease in insulin stores, suggesting that palmitate inhibits the glucose-stimulated increase in preproinsulin gene expression. The mechanisms by which palmitate affects preproinsulin gene expression implicate both preproinsulin mRNA stability and transcription, as suggested by an actinomycin D decay assay, quantification of primary preproinsulin transcripts, and transient transfection experiments in Min6 cells. Metabolism of palmitate is not required to obtain these effects, inasmuch as they can be reproduced by 2-bromopalmitate. However, oleate and linoleate did not significantly influence preproinsulin mRNA levels. We conclude that insulin release and preproinsulin gene expression are not coordinately regulated by palmitate and that chronically elevated FFA levels may interfere with beta-cell function and be implicated in the development of noninsulin-dependent diabetes.

Animals↗
PubMed1999

Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells.

Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A>>C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K+-stimulated secretion of dense core vesicles, indicating that, in beta-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxyterminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.

Animals↗
PubMed1998

Enhanced secretion of amylase from exocrine pancreas of connexin32-deficient mice.

To determine whether junctional communication between pancreatic acinar cells contributes to their secretory function in vivo, we have compared wild-type mice, which express the gap junctional proteins connexin32 (Cx32) and connexin26, to mice deficient for the Cx32 gene. Pancreatic acinar cells from Cx32 (-/-) mice failed to express Cx32 as evidenced by reverse transcription-PCR and immunolabeling and showed a marked reduction (4.8- and 25-fold, respectively) in the number and size of gap junctions. Dye transfer studies showed that the extent of intercellular communication was inhibited in Cx32 (-/-) acini. However, electrical coupling was detected by dual patch clamp recording in Cx32 (-/-) acinar cell pairs. Although wild-type and Cx32 (-/-) acini were similarly stimulated to release amylase by carbamylcholine, Cx32 (-/-) acini showed a twofold increase of their basal secretion. This effect was caused by an increase in the proportion of secreting acini, as detected with a reverse hemolytic plaque assay. Blood measurements further revealed that Cx32 (-/-) mice had elevated basal levels of circulating amylase. The results, which demonstrate an inverse relationship between the extent of acinar cell coupling and basal amylase secretion in vivo, support the view that the physiological recruitment of secretory acinar cells is regulated by gap junction mediated intercellular communication.

Amylases↗
PubMed1998

Upregulation of connexin 26 between keratinocytes of psoriatic lesions.

To assess whether the expression of connexins (Cx) by keratinocytes is altered under conditions of abnormal epidermal differentiation, we have compared Cx26, Cx32, Cx37, Cx40, and Cx43 in the epidermis of 11 psoriatic patients who had not been treated for at least 1 mo and of seven healthy individuals. In all samples of fully mature psoriatic plaques, we have observed a massive expression of Cx26, as judged at both the transcript level (northern blot) and the protein level (immunofluorescence). This protein became consistently detected between keratinocytes of the basal and granular layers at the periphery of psoriatic plaques and in all layers of fully developed psoriatic epidermis, except in regions of parakeratosis. None or a minimal amount of Cx26 was observed in both control and nonlesional regions of psoriatic epidermis. Psoriatic plaques also contained Cx43, the prominent gap junction protein in the interfollicular epidermis of normal human skin. The levels of this protein appeared to be slightly higher in psoriatic than in control skin, as judged at both the transcript level (northern blot) and the protein level (immunofluorescence). Three other connexins (Cx32, Cx37, and Cx40), which are not observed in control interfollicular epidermis, were not induced in either nonlesional or lesional regions of psoriatic skin. The data indicate that selective changes in the normal expression of connexins by keratinocytes are associated with the changes in the proliferation and differentiation program that these cells undergo in psoriasis.

Adult↗
PubMed1998

The distribution of murine 115-kDa epithelial microtubule-associated protein (E-MAP-115) during embryogenesis and in adult organs suggests a role in epithelial polarization and differentiation.

In interphase cells microtubules play fundamental roles in the intracellular distribution and movement of organelles and vesicles and thereby contribute to cellular polarization and differentiation. The organization of microtubules varies with the cell type and is presumably controlled by tissue-specific microtubule-associated proteins (MAPs). The 115-kDa epithelial MAP (E-MAP-115) has been identified as a microtubule-stabilizing protein predominantly expressed in cell lines of epithelial origin. To assess a putative function of E-MAP-115 in epithelial morphogenesis in vivo, we have cloned the cDNA encoding the murine protein and studied the cellular distribution of E-MAP-115 mRNA and protein during murine embryogenesis and in adult organs. Analysis of the predicted amino acid sequence of murine E-MAP-115 revealed 81% sequence identity with its human homolog, the best-conserved part of the protein being the microtubule-binding site. Our data indicate that E-MAP-115 is expressed in several epithelia from 9.5 days of embryogenesis onwards and that its expression levels increase during development. From 14.5 days onwards, E-MAP-115 mRNA is found in some neuronal cells as well. In adult organs, E-MAP-115 is most abundant in epithelial cells of kidney tubules, in absorptive cells of the intestine and is widely distributed in the testis. E-MAP-115 expression correlates with the differentiation of certain epithelial cell types: in the adult intestine, for example, E-MAP-115 mRNA and protein are more abundant in the differentiating than in the proliferative cell compartment. Moreover, E-MAP-115 expression clearly correlates with the degree of cellular apicobasal polarity. In the developing kidney, E-MAP-115 mRNA is detected in the cuboidal cells of S-shaped bodies, of primitive tubules and glomerula, whereas, E-MAP-115 mRNA and protein are absent from mature podocytes which have lost their initial apico-basal polarity. The pattern of distribution of E-MAP-115 in vivo is so far unique for a MAP. Taken together, our results provide support for a role of E-MAP-115 in reorganizing the microtubule cytoskeleton during epithelial cell polarization and differentiation.

Aging↗
PubMed1998

Insulin, but not glucose lowering corrects the hyperglucagonemia and increased proglucagon messenger ribonucleic acid levels observed in insulinopenic diabetes.

The factors that regulate glucagon biosynthesis and proglucagon gene expression are poorly defined. We previously reported that insulin inhibits proglucagon gene expression in vitro. In vivo, however, the effects of insulin on the regulation of the proglucagon gene have been controversial. Furthermore, whether glucose plays any role alone or in conjunction with insulin on proglucagon gene expression is unknown. We investigated the consequences of insulinopenic diabetes on glucagon gene expression in the endocrine pancreas and intestine and whether insulin and/or glucose could correct the observed abnormalities. We show here that in the first 3 days after induction of hyperglycemia by streptozotocin, rats have levels of plasma glucagon and proglucagon messenger RNA comparable to those of normoglycemic controls despite hyperglycemia. With more prolonged diabetes, plasma glucagon and proglucagon messenger RNA levels increase; this increase is corrected by insulin treatment, but not by phloridzin despite normalization of the glycemia by both treatments. Proglucagon gene expression exhibits the same regulatory response to glucose and insulin in both pancreas and ileum. We conclude that insulin tonically inhibits proglucagon gene expression in the pancreas and ileum and that glucose plays a minor, if any, role in this regulation.

Animals↗
PubMed1997

Hypertension increases connexin43 in a tissue-specific manner.

BACKGROUND: Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. METHODS AND RESULTS: Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart. CONCLUSIONS: The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Animals↗