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P Meda

Publications and source records attributed to P Meda.

At least 55 records · Page 3Linked to original sources
PubMed1997

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Animals↗
PubMed1997

In vivo modulation of connexins 43 and 26 of human epidermis by topical retinoic acid treatment.

After 14 weeks of topical application of 0.1% all-trans-retinoic acid to the napes of volunteers, we observed a 2.5-fold increase in the thickness of epidermis, owing to an increase (p < 0.001) in the number and size of keratinocytes and the induction of keratin 6. These changes in the differentiation of epidermal keratinocytes were paralleled by an increase in the amount of Cx43, a connexin that is normally expressed in human epidermis, and by the massive induction of Cx26, which is barely detectable in normal interfollicular epidermis, as judged at both the transcript (Northern blotting) and the protein level (immunolabeling). In contrast, retinoic acid treatment did not alter the morphology and connexin pattern of hair follicles or of sebaceous and sweat glands, and did not induce the expression of other connexins (C32, Cx37, Cx40) in either skin adnexae or epidermis. These observations suggest that the expression of two distinct connexins by interfollicular keratinocytes is related to selective changes in the differentiation program of epidermis that are induced by retinoic acid.

Administration, Topical↗
PubMed1997

Hyperinsulinemia-induced hypoglycemia is enhanced by overexpression of connexin 43.

To assess whether cell to cell communications via connexins (Cx) participate to insulin secretion in vivo, we studied insulinoma cells (INS1) implanted in rats after stable transfection with connexin 43 (Cx43). We found that compared to wild-type and transfected cells, which in vivo express modest levels of Cx43 and junctional communication, cells overexpressing Cx43 communicated extensively, featured decreased growth, and induced a much higher hyperinsulinemia. As a result, rats with insulinomas made of these cells became more severely hypoglycemic than rats implanted with either wild-type, neomycin-transfected cells or cells transfected with a Cx43 antisense complementary DNA. Rats implanted with transfected cells that expressed modest level of Cx43 showed levels of circulating insulin similar to those in rats implanted with wild-type INS1 cells. The data show that overexpression of Cx43 influences the growth and secretion of the implanted insulinoma cells, providing evidence for a contribution of Cx-mediated cell to cell communication in the functioning of insulin-producing cells in vivo.

Animals↗
PubMed1996

Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis.

Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis.

Botulinum Toxins↗
PubMed1996

The caudal-related homeodomain protein Cdx-2/3 regulates glucagon gene expression in islet cells.

Glucagon gene transcription in the endocrine pancreas is regulated by at least four cis-acting DNA control elements. We showed previously that G1 is critical for alpha cell-specific expression. G1 contains three AT-rich sequences important for promoter function, which represent candidate binding sites for homeodomain transcription factors. Performing reverse transcription-polymerase chain reaction amplifications with degenerate oligonucleotide primers homologous to the Antennapedia homeobox, cDNA clones corresponding to the caudal-related gene cdx-2/3 were predominantly obtained from glucagon-producing cells and primary non-beta cells. From RNase protection and polymerase chain reaction analyses, cdx-2/3 turned out to be the only caudal-related gene that is expressed at significant levels in cells of the endocrine pancreas. Cdx-2/3 binds with high affinity to an AT-rich motif of G1, which matches the consensus binding site of caudal-related proteins. In the glucagon-producing hamster cell line InR1G9, Cdx-2/3 is a subunit of complex B3 formed on G1. Alternative splicing generates two cdx-2/3 transcripts in islet cells, coding for a full-length protein and an amino-terminally truncated isoform. Although both isoforms bind G1 with similar affinity, only the full-length Cdx-2/3 A protein activates glucagon gene transcription in non-glucagon-producing cells, transcriptional activation being dose-dependent. We therefore conclude that the caudal-related gene cdx-2/3 is implicated in the transcriptional control of glucagon gene expression in the alpha cells of the islets of Langerhans.

Alternative Splicing↗
PubMed1996

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

3-O-Methylglucose↗
PubMed1996

The role of gap junction membrane channels in secretion and hormonal action.

Connexins, gap junctions, and coupling are obligatory features of both endocrine and exocrine glandular epithelia. Evidence from these two types of tissues, and particularly from pancreatic islets and acini, indicates that cell-to-cell communication via gap junction channels is required for proper biosynthesis, storage, and release of specific secretory products. However, endocrine and exocrine glands express a different set of connexins and show opposite connexin and coupling changes in relation with the activation and inhibition of their secretory function. Also, several hormones modulate connexin and coupling expression, and junctional coupling affects hormonal stimulation. These observations indicate that gap junction channels play an important role in the control of secretion and hormonal action.

Cell Degranulation↗
PubMed1996

Gap junction involvement in secretion: the pancreas experience.

1. Gap junctions and junction-mediated cell-to-cell communications are obligatory features of gland cells, whatever their secretory product is. 2. Studies on pancreatic islets and acinar cells indicate that cell-to-cell communication via gap junction channels is required for proper biosynthesis, storage and release of both insulin and amylase. 3. However, the endocrine and exocrine portions of the pancreas show opposite connexin (Cx) and coupling changes in relation to the activation and inhibition of their secretory functions. 4. These differences may be accounted for by the expression of Cx43 in pancreatic islets and of Cx26 and Cx32 in pancreatic acini. This alternative expression of connexin isoforms is also found in several other endocrine and exocrine glands. 5. These observations indicate that connexin-made channels play a central role in the control of secretory events.

Animals↗
PubMed1996

[Role of intercellular communication via gap junctions in insulin secretion].

Alike most other cell types of eukaryotic organisms, the insulin-producing beta-cells of pancreatic islets are functionally interconnected by specialized regions of their cell membrane, referred to as gap junctions. These structures represent concentrations of channels which are formed by the proteins of the connexin family, and that are specialized for the exchange between neighboring cells of cytoplasmic ions and small molecules. This exchange is referred to as junctional coupling. Increasing evidence indicates that a normal expression of connexins and junctional coupling is essential to ensure proper insulin biosynthesis and secretion.

Connexin 43↗
PubMed1995

Adequate connexin-mediated coupling is required for proper insulin production.

To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.

Animals↗
PubMed1995

Heterogeneous secretion of individual B cells in response to D-glucose and to nonglucidic nutrient secretagogues.

We used a hemolytic plaque assay for insulin to determine whether the same pancreatic B cells respond to D-glucose, 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and the association of this nonmetabolized analogue of L-leucine with either the monomethyl ester of succinic acid (SME) or the dimethyl ester of L-glutamic acid (GME). During a 30-min incubation in the absence of D-glucose, BCH alone (5 mM) had no effect on insulin release. In contrast, the combination of BCH with either SME (10 mM) or GME (3 mM) stimulated insulin release to the same extent observed in the sole presence of 16.7 mM D-glucose. The effects of BCH plus SME and BCH plus GME on both percentage of secreting B cells and total insulin output were little affected in the presence of D-glucose concentrations ranging from 0 to 16.7 mM. Varying the concentration of SME from 2 to 10 mM also did not influence these effects. In other experiments, the very same B cells were first exposed 45 min to 16.7 mM D-glucose, then incubated 45 min in the presence of only BCH and SME. Under these conditions, most (80.3 +/- 2.5%) of the cells contributing to insulin release did so during both incubation periods. Furthermore, virtually all cells responding to BCH and SME during the second incubation corresponded to cells also responsive to D-glucose during the first incubation. Similar observations were made when the sequence of the two incubations was reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1994

Heterogeneity and contact-dependent regulation of amylase release by individual acinar cells.

We have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose-dependent manner, during a 30-min exposure to concentrations of carbamylcholine (CCh) ranging from 10(-8) to 10(-5) M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10(-8) - 10(-7) M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10(-6) - 10(-5) M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact-dependent effect, we found that 10(-3) M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact-dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion.

Amylases↗
PubMed1994

Effects of vitamin A deficiency and repletion on rat glucagon secretion.

To determine whether vitamin A is involved in pancreatic alpha cell function, we tested for (a) effects of vitamin A deficiency on glucagon release from perifused islets and perfused pancreases, and (b) the presence of cytosolic retinol-binding proteins (CRBP) and retinoic acid-binding proteins (CRABP), in the glucagon-secreting alpha cell line, ln-R1-G9. Arginine 19 mM plus glucose 2.8 mM-stimulated glucagon secretion was markedly impaired in islets and pancreases of vitamin A-deficient rats or rats that had at some time been cycled through vitamin A deficiency (ever A-def) despite repletion with retinoids for 2-4 weeks. Insulin secretion was impaired likewise. Repletion starting early in the development of vitamin A deficiency and for a longer period of time (18 or 60 days) did not restore glucagon secretion, but did normalize insulin secretion. CRBP and CRABP were present in ln-R1-G9 cells. We conclude that (a) vitamin A deficiency is associated with a defect in glucagon secretion; (b) The defect in secretion occurs early in the course of vitamin A deficiency; (c) The defect persists despite repletion; and (d) The requirement of vitamin A for secretion and the presence of CRBP and CRABP in glucagon-secreting cells support a physiologic role for vitamin A at the alpha cell level.

Animals↗
PubMed1994

Topography of mammalian connexins in human skin.

We have explored the expression of gap junction proteins in normal human skin by immunostaining cryostat sections (indirect immunofluorescence) or lyophilized epidermis (Western blotting) with antibodies against four mammalian connexins Cx26, Cx32, Cx40, Cx43; and by hybridizing total epidermal RNA (Northern blotting) with cRNA probes for Cx26, Cx32, and Cx43. We found that epidermal keratinocytes express Cx43 but not Cx26, Cx32, or Cx40. This expression was minimal in the basal layer, much higher in the spinous layer, reduced in the granular layer, and absent in the stratum corneum. Immunostaining for Cx43 was also observed in sebaceous glands, hairs, and eccrine sweat ducts. The two latter epidermal adnexae were also markedly labeled by antibodies against Cx26, a gap junction protein that was undetectable by immunofluorescence in interfollicular keratinocytes. Immunoblots of polyacrylamide gel electrophoresis-separated epidermal proteins and hybridization of epidermal RNA confirmed the presence of Cx43 in epidermis. These observations indicate that 1) Cx43 and Cx26 are components of human keratinocyte gap junctions; 2) these two proteins are differentially expressed in the interfollicular epidermis and the skin adnexae; 3) in interfollicular epidermis, Cx43 is a predominant gap junction protein, mostly expressed by the differentiating spinous cells; 4) Cx43 distribution is in accordance with the extensive dye coupling previously observed in this epidermal compartment.

Animals↗
PubMed1994

[Ca2+]i and protein kinase C in vasopressin-induced prostacyclin and ANP release in rat cardiomyocytes.

Exposure of cultured, spontaneously beating rat cardiomyocytes to arginine vasopressin (AVP) led to marked increases in the release of prostacyclin (PGI2) and atrial natriuretic peptide (ANP). These responses were accompanied by a rapid, transient rise of cytosolic free Ca2+ concentration ([Ca2+]i) and of membranous protein kinase C (PKC) activity. Ca2+ influx and PKC activity appeared to play important but distinct roles in AVP-induced cellular responses, insofar as only AVP-induced ANP secretion was abolished by the Ca2+ channel antagonist nifedipine, whereas both AVP-induced PGI2 production and ANP release were abolished by the PKC inhibitors staurosporine and CGP-41251. The AVP-induced increase in [Ca2+]i could also be mimicked with the vasopressin (V1-subtype) agonist Octapressin, but not with the V2-agonist 1-desamino-8-D-arginine vasopressin, and was fully abolished by the V1-antagonist [d(CH2)5Tyr(Me)]AVP, but not by d(CH2)5-D-Leu-VAVP (V1-/V2-antagonist). These results indicate that V1-vasopressinergic receptors mediate AVP-induced PGI2 production and ANP secretion in rat cardiomyocytes and that, whereas both Ca2+ influx and PKC activation are required for AVP-induced ANP secretion, AVP-induced PGI2 formation is mainly regulated by PKC.

Alkaloids↗
PubMed1994

Insulin gene transcription is decreased rapidly by lowering glucose concentrations in rat islet cells.

Glucose is the main physiological regulator of insulin biosynthesis and secretion. To exert an effect on biosynthesis, stimulatory concentrations of glucose act on insulin gene expression at both transcriptional and posttranscriptional levels. The relative importance of these two effects is controversial. Studies on the effect of decreasing glucose concentrations in vitro have been contradictory as to its rapidity and magnitude. We have investigated whether a decrease in glucose concentrations from a stimulatory (11 mM) to a nonstimulatory (2 mM) level regulates insulin gene expression in both isolated rat islets and INS-1 cells, an insulinoma-derived cell line that retains a number of the differentiation characteristics of native beta-cells. We show that, in the presence of a nonstimulatory concentration of glucose, insulin mRNA levels decrease rapidly in both rat islets and INS-1 cells. Low glucose decreases insulin gene expression at the transcriptional level, and this effect is mediated by DNA sequences present within 254 base pairs of the 5'-flank of the rat insulin I gene. We conclude that lowering glucose from 11 to 2 mM decreases insulin mRNA rapidly by a transcriptional effect.

Animals↗
PubMed1993

Effects of anions and/or cell volume on the permeance of an apical water pathway induced by Hg in toad skin epithelium.

Hg compounds block membrane transport units behaving as water channels. Here we show that Hg induces an apical water pathway in toad skins pretreated with 10(-3) M CH3ClHg or HgCl2, added to the outer bathing medium. Washing with SO4-Ringer caused a several-fold increase in net water flow (Jw) and osmotic permeability coefficient (Pf) that was reversed by re-exposure to Cl- or NO3-Ringer and mimicked by gluconate-Ringer. These Pf changes could be elicited repeatedly and were present if, and only if, anion replacements took place in the inner bathing solution. Such inner polarity was related to the anion permeability of the epidermal basolateral membrane: impermeant anions (SO4, gluconate) increased Pf; permeant anions (Cl, NO3) did not change basal Pf but reversed the high Pf induced by impermeant anions. Hg induced the appearance of aggregates that persisted despite repeated washings of the skins during 4-5 h, and whether Pf was high (SO4-Ringer) or low (Cl-Ringer) before skin fixation. The Hg-induced apical water pathway in toad skin appears to be a unique model for studying the interplay between cell volume, cell ionic composition and water permeability.

Animals↗