Biomedical subjects

C Montrose-Rafizadeh

Publications and source records attributed to C Montrose-Rafizadeh.

35 records · Page 2Linked to original sources

PubMed1996

GIP regulates glucose transporters, hexokinases, and glucose-induced insulin secretion in RIN 1046-38 cells.

Acute studies of glucose-dependent insulinotropic peptide (GIP) have shown that GIP can synergize with glucose in stimulating insulin secretion both in vivo and in vitro. Here we studied the effects of extended exposure of RIN 1046-38 cells, an insulin-secreting cell line, to GIP and the mechanisms by which GIP synergizes with glucose in stimulating insulin secretion. Incubation of the cells with 100 nM GIP in the presence of glucose for 12 h significantly increased insulin release (287 +/- 31.7 vs. 102 +/- 9.7 ng/mg protein; n = 3), intracellular insulin content (12.8 +/- 0.83 vs. 8.2 +/- 0.52 ng/mg protein; n = 3), and insulin mRNA (approximately 2.7-fold; 24 h incubation) when compared to cells cultured with glucose alone. The insulinotropic effects of GIP on RIN 1046-38 cells were accompanied by an up-regulation of GLUT-1 and hexokinase I mRNA (1.75-fold) compared to non-GIP-treated cells; mRNA levels of GLUT-2 and glucokinase were unchanged by GIP, in the presence or absence of glucose. Our study suggests that the mechanism by which extended exposure of RIN 1046-38 cells to GIP increases glucose-stimulated insulin secretion includes up-regulation of glucose sensing elements.

Animals↗

PubMed1996

Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix.

Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glucagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, beta cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 +/- 2.5 vs. 37.1 +/- 2.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single beta cell was reduced by 40-60% (p < 0.02) compared to that released from isolated beta cells derived from a 3-day culture of islets. Finally, there was a 35-55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal beta-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.

Animals↗

PubMed1995

Cloning of a putative human voltage-gated chloride channel (CIC-2) cDNA widely expressed in human tissues.

We have cloned a cDNA from the human epithelial cell line T84 whose predicted amino acid sequence shows 93.9% identity with rat CIC-2. Mapping by somatic cell hybrids and polymerase chain reaction localizes the gene corresponding to this cDNA to chromosome 3q26-qter. The major transcription start site assessed by RNA primer extension is 100 nt upstream of the putative translation initiation codon. Analysis of the 5' flanking sequence revealed a high GC content and lack of common transcriptional elements such as TATA and CCAAT boxes. Northern blot analysis indicated wide organ distribution including tissues affected in cystic fibrosis (CF) and expression in an airway epithelial cell line derived from a CF patient. The high degree of sequence similarity and similar tissue distribution to rat CIC-2 suggests that this cDNA encodes the human CIC-2 voltage-gated chloride channel. Since this chloride channel is present in epithelial tissues it may be amenable to manipulation to circumvent the chloride secretion defect observed in CF.

Amino Acid Sequence↗

PubMed1995

Glucagon-like peptide-1 affects gene transcription and messenger ribonucleic acid stability of components of the insulin secretory system in RIN 1046-38 cells.

It has been previously demonstrated that the enteric hormone glucagon-like peptide-1 (7-36 amide) (GLP-1) has acute effects on glucose-induced insulin secretion by RIN 1046-38 cells. In this study, we investigated the effects of extended exposure of RIN 1046-38 cells to GLP-1 and examine the mechanism by which GLP-1 synergizes with glucose in stimulating insulin secretion. Compared with cells cultured with glucose alone, incubation of cells with glucose plus 1 or 10 nM GLP-1 for 12 or 24 h significantly increased insulin release by about 3-fold, intracellular insulin content by 1.5-fold, and insulin messenger RNA (mRNA) by almost 2.5-fold. The insulinotropic effects of GLP-1 on RIN 1046-38 cells were accompanied by an up-regulation of both glucose transporter-1 (GLUT-1) and hexokinase I mRNA by about 2-fold. mRNA levels of GLUT-2 and glucokinase, which were low in controls, were unchanged by GLP-1 treatment. Treatment of cells with a transcription inhibitor, actinomycin D, demonstrated that elevated insulin mRNA levels after a GLP-1 exposure are mainly due to stabilization of the mRNA. In contrast, the elevated mRNA levels of GLUT-1 and hexokinase I are the result of increased transcription stimulated by GLP-1 exposure. Actinomycin D blunted the GLP-1 effect on insulin release but did not affect GLP-1 mediated elevation of insulin mRNA. This suggests that actinomycin D inhibits the transcription of the proteins necessary for insulin biosynthesis and insulin release, such as GLUT-1 and hexokinase I. Our study suggests that the mechanisms by which extended exposure of RIN 1046-38 cells to GLP-1 increases glucose-stimulated insulin secretion include significant up-regulation of glucose-sensing elements.

Animals↗

PubMed1994

Incretin hormones regulate glucose-dependent insulin secretion in RIN 1046-38 cells: mechanisms of action.

Glucagon-like peptide-1-(7-36) amide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are known incretin hormones, released from enteroendocrine cells in response to food, that enhance insulin secretion, but only in the presence of elevated blood glucose. We used a rat insulinoma cell line, RIN 1046-38, to study the mechanisms underlying the interaction of incretins and glucose. We measured insulin secretion using RIA and the reverse hemolytic plaque assay. GLP-1 stimulates insulin secretion, with a half-maximal concentration of 34 pM. GLP-1 is approximately 2 orders of magnitude more potent than GIP. GLP-1 and GIP have additive effects at submaximal concentrations, but probably not at maximal concentrations, suggesting a common signal transduction pathway. The glucose requirement for GLP-1 action can be replaced by cell membrane depolarization (20 mM KCl in the extracellular medium), suggesting that a rise of intracellular Ca2+ may be an early step required for GLP-1 action. GLP-1 stimulates insulin secretion by significantly increasing the maximum rate of insulin secretion from 10.3 +/- 2.25 to 25.2 +/- 2.94 ng insulin/mg protein.h. GLP-1 acts by recruiting 1.5-fold more cells to secrete insulin as well as enhancing insulin secretion by individual cells. Combinations of stimuli, such as glucose, cell membrane depolarization, and GLP-1, can recruit 90% of RIN 1046-38 cells to secrete insulin.

Animals↗

PubMed1994

Glucagon-like peptide-1(7-36) amide (GLP-1) enhances insulin-stimulated glucose metabolism in 3T3-L1 adipocytes: one of several potential extrapancreatic sites of GLP-1 action.

We investigated the effects of glucagon-like peptide-1(7-36) amide, GLP-1, on glucose metabolism in 3T3-L1 adipocytes and we used polymerase chain reaction to search for the presence of GLP-1 receptors in various rat tissues. GLP-1 at 1 nM significantly increased insulin-mediated 2-deoxyglucose uptake by 40% while having no effect on basal uptake. In conjunction with the elevated uptake, the insulin-dependent incorporation of 14C-glucose into fatty acids was also increased. Moreover, neither glycogen synthesis nor insulin binding to its receptor were affected by GLP-1. In addition to the presence of GLP-1 receptor in pancreas we found messenger RNA for this receptor in brain, kidney, heart, fat, skeletal muscle, liver, and intestine. This study indicates that GLP-1, in addition to its well known effect of stimulating insulin secretion, may improve insulin responsiveness by promoting fatty acid synthesis in adipose cells and possibly modulating insulin signaling in other insulin sensitive tissues.

3T3 Cells↗

PubMed1992

Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gene transcription and alternative RNA splicing in a model of developing intestinal epithelium.

Transcriptional and post-transcriptional regulation of CFTR (cystic fibrosis transmembrane conductance regulator) gene expression was studied in HT29 cells. It is known that the abundance of CFTR mRNA increases during differentiation of pluripotent HT29-18 cells and is maintained at high levels in the stably differentiated HT29-18-C1 subclone. Nuclear run-on assays suggest that increased transcription of the CFTR gene explains the increased abundance of total CFTR mRNA in differentiated HT29 cells. The increased transcription cannot be ascribed to cell cycle-dependent expression of the CFTR gene or to changes in CFTR gene copy number between subcloned cells. Similar to native tissue cells, differentiated HT29 cells contain low copy numbers of CFTR transcripts (1-5/cell), and a portion of the CFTR transcripts are alternatively spliced to remove exon 9 (and make 9-mRNA). During differentiation of HT29-18 cells, the absolute amount of full-length CFTR mRNA increases 8-fold, whereas the amount of 9- mRNA increases 18-fold. The fraction of 9- mRNA in the CFTR mRNA pool is increased in differentiated HT29 cells. The results show that gene transcription regulates the abundance of CFTR transcripts and that regulatory control of alternative RNA splicing may also be a cellular mechanism to modulate CFTR function.

Base Sequence↗

PubMed1992

Effect of hypoxia on endothelin-1 production by pulmonary vascular endothelial cells.

Endothelin-1 (ET-1), a peptide product of endothelial cells, is mitogenic for fibroblasts and smooth muscle cells. In this study we examined the effect of hypoxia on ET-1 production by bovine pulmonary vascular endothelial cells. Bovine pulmonary artery (BPAE) and microvascular endothelial (BMVE) cells were isolated, grown in tissue culture, and characterized by the presence of Factor VIII related antigen and LDL uptake. Baseline production of ET-1 by BPAE cells (measured by radioimmunoassay) increased over time. BMVE cells produced one tenth the amount of ET-1 as produced by the pulmonary artery endothelial cells under the same conditions. In both cell types, hypoxia (0% O2) significantly reduced the amount of ET-1 at 48 h. Restoration of normoxia in 21% O2 for 48 h resulted in a return of ET-1 levels to baseline. Northern blot analysis showed decreased ET-1 mRNA in cells exposed to hypoxia for 48 h. These data demonstrate that pulmonary vascular endothelial cells respond to hypoxia by reversibly decreasing ET-1 production, and this attenuation is likely regulated at the level of transcription.

Animals↗

PubMed1991

Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

Amino Acid Sequence↗

PubMed1991

Cellular differentiation regulates expression of Cl- transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells.

The gene defective in cystic fibrosis has recently been shown to code for a membrane protein designated the "cystic fibrosis transmembrane conductance regulator" (CFTR) protein. While it has been shown that detectable levels of the mRNA for the normal CFTR protein are present in epithelial cells from different tissues, factors which regulate CFTR expression have not been identified. A clonal cell line originating from a human colon adenocarcinoma (HT29-18) differentiates to multiple epithelial cell types when deprived of glucose in the culture medium. In these studies, mRNA isolated from these cells was examined by hybridization to a 1.45-kilobase cDNA probe which encodes transmembrane portions of the CFTR protein between exons 13 and 19. Cellular differentiation of HT29-18 causes a 9-18-fold increase in CFTR mRNA abundance versus the mRNA for the structural proteins actin and tubulin. Cellular differentiation also causes a 5-fold increase in second messenger-regulated Cl- transport which is sensitive to a Cl- channel blocker (diphenylamine 2-carboxylate). Subclones of HT29-18 which are committed to differentiate to either a mucin-secreting (HT29-18-N2) or an "enterocyte-like" (HT29-18-C1) phenotype have also been examined. In both subclones, elevated levels of CFTR mRNA are observed when compared with undifferentiated HT29-18 cells. However, during cellular differentiation, the regulation of CFTR mRNA abundance and membrane enzyme expression by the subclones is different from HT29-18. The results show that elevated CFTR mRNA occurs in multiple differentiated intestinal epithelial cell types, despite a phenotype-specific regulation of membrane protein expression. This suggests that CFTR expression plays a role in the differentiated functions of multiple epithelial phenotypes and that both cellular differentiation and cellular phenotypes are factors which regulate CFTR expression.

Biological Transport↗

PubMed1991

Role of intracellular calcium in volume regulation by rabbit medullary thick ascending limb cells.

Previous studies demonstrated that Ca2(+)-activated K+ channels in luminal membrane of rabbit medullary thick ascending limb cells (MTAL) are activated on exposure of the cells to hyposmotic solutions [J. Taniguchi and W. B. Guggino. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F347-F352, 1989]. In this study, we investigated the mechanism of activation of Ca2(+)-activated K+ channels in MTAL cells exposed to hyposmotic solutions. MTAL cells swell in hyposmotic medium and regulate volume back toward the starting volume. This regulatory volume decrease (RVD) is inhibited at high medium K+ concentrations or by presence of quinine or Ba2+ in extracellular medium, suggesting involvement of K+ channels. Measurements of intracellular Ca2+ concentrations with fura-2 show that intracellular Ca2+ rises in hyposmotic solutions and that this rise does not occur in absence of extracellular Ca2+. Nifedipine and verapamil also inhibit rise in intracellular Ca2+. Decreasing intracellular Ca2+ by removal of external Ca2+ in presence of EDTA or by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibits RVD. We conclude that hypotonic solutions activate K+ efflux probably via K+ channels and Ca2+ influx via a nifedipine- and verapamil-sensitive pathway. Lowering intracellular Ca2+ removes the ability of MTAL cells to regulate volume in hyposmotic solutions.

Animals↗

PubMed1991

Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis.

Cystic fibrosis (CF) is the most common, lethal inherited disorder in the Caucasian population. We have recently reported two African-American patients with nonsense mutations in each CF gene and severe pancreatic disease, but mild pulmonary disease. In order to examine the effect of these nonsense mutations on CF gene expression, bronchial and nasal epithelial cells were obtained from one of these patients (no. 246), a compound heterozygote for nonsense mutations R553X and W1316X; a healthy normal individual; a patient (no. 528) homozygous for the common CF mutation (delta F508); and a CF patient (no. 272) who carries the R553X mutation and a missense mutation, S549N. When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by polymerase chain reaction using primers derived from the CF gene, DNA fragments of the predicted size were observed. However, patient no. 246 with nonsense mutations in each CF gene has no detectable cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA, and therefore should have severely diminished, and possibly absent, CFTR protein. Furthermore, less than 2% of the CFTR transcripts in nasal epithelial cells from patient no. 272 (S549N/R553X) were derived from the gene with the nonsense mutation. We conclude that severe reduction in CFTR mRNA causes CF, but can have different consequences in the lung and pancreas.

Adolescent↗

PubMed1990

A delayed rectifier potassium current in Xenopus oocytes.

A delayed voltage-dependent K+ current endogenous to Xenopus oocytes has been investigated by the voltage-clamp technique. Both activation and inactivation of the K+ current are voltage-dependent processes. The K+ currents were activated when membrane potential was depolarized from a holding potential of -90 to -50 mV. The peak current was reached within 150 ms at membrane potential of +30 mV. Voltage-dependent inactivation of the current was observed by depolarizing the membrane potential from -50 to 0 mV at 10-mV increments. Voltage-dependent inactivation was a slow process with a time constant of 16.5 s at -10 mV. Removal of Ca2+ from the bath has no effect on current amplitudes, which indicates that the current is Ca2+)-insensitive. Tail current analysis showed that reversal potentials were shifted by changing external K+ concentration, as would be expected for a K(+)-selective channel. The current was sensitive to quinine, a K+ channel blocker, with a Ki of 35 microM. The blockade of quinine is voltage-independent in the range of -20 to +60 mV. Whereas oocytes from the same animal have a relatively homogeneous current distribution, average amplitude of the K+ current varied among oocytes from different animals from 30 to 400 nA at membrane potential of +30 mV. Our results indicate the presence of the endogenous K+ current in Xenopus oocytes with characteristics of the delayed rectifier found in some nerve and muscle cells.

Animals↗

PubMed1990

Cell volume regulation in the nephron.

Nearly every cell in the kidney can volume regulate in response to a hypertonic challenge. Some are able to respond immediately to hyperosmotic media by a RVI. Other cells require stimulation prior to exposure to hyperosmolarity to demonstrate RVI. An increase of intracellular osmolytes during RVI usually occurs by an increase of NaCl influx either via the activation of parallel Na(+)-H+ and Cl(-)-HCO3- exchangers, or Na(+)-K(+)-2Cl- cotransporters. Medullary and papillary cells use organic solutes as well to increase the intracellular concentration of osmolytes. In response to a hypotonic challenge, a RVD response has been demonstrated in the majority of the kidney cells. The efflux of solute during RVD is usually via K+ loss by activation of conductance pathways. Stretch-activated K+ channels and Ca2(+)-activated-K+ channels have been shown to be stimulated in cells exposed in hyposmotic solutions and could thus be involved in RVD. The accompanying anion loss is less well-defined but could be either Cl- or HCO3- in different segments of the nephron. In some cells, the reduction of intracellular solute content is via an efflux of organic osmolytes. Thus it appears that cells in all segments of the nephron volume regulate in response to osmotic stresses. This regulation may be an essential part of transepithelial transport since the cells have to survive transcellular fluxes of osmolytes. It may be particularly important in the kidney to control cell volume both in response to changes in osmolarity and transcellular fluxes of solute in order to maintain proper flow of fluid through the nephron.

Animals↗

PubMed1990

Transport of urate and p-aminohippurate in rabbit renal brush-border membranes.

The mechanisms involved in urate and p-aminohippurate (PAH) transport in the rabbit renal brush-border membrane were investigated through study of membrane vesicles. Transport of [14C]urate and [3H]PAH was measured by a rapid filtration method. As previously reported by others, no OH(-)-PAH exchanger could be demonstrated by imposing an outwardly directed OH- gradient (pHin 7.4, pHout 6). In contrast, an OH(-)-lactate exchanger (or H(+)-lactate cotransport) was demonstrated. In the presence of valinomycin and an inwardly directed K+ gradient, both [14C]urate and [3H]PAH vesicle uptake were stimulated, demonstrating a potential-driven transport of these two anions. Probenecid, PAH, or cold urate decreased potential-driven urate uptake, suggesting that this transport was facilitated by a specific transport mechanism. The potential-driven urate transport described here may play a role in the second step of urate secretion in rabbits, because rate (or PAH) is transported across the brush-border membrane from the negative interior of the cell to the more positive omen.

Aminohippuric Acids↗

PubMed1989

Carrier-mediated transport of tetraethylammonium across rabbit renal basolateral membrane.

Mechanisms of tetraethylammonium (TEA) transport were investigated in basolateral membrane (BLM) vesicles from rabbit renal cortex. Preloading vesicles with 10 mM TEA or 1 mM mepiperphenidol stimulated the 15-s uptake of [14C]-TEA compared with control vesicles (258 and 200%, respectively) and produced an overshoot of the equilibrium value (3 and 1.6 times, respectively). In the presence of a K+ gradient, net TEA uptake was also increased (and showed an overshoot of 2-fold) when the membrane potential of vesicles was made interior negative by adding valinomycin. Both TEA-TEA exchange and the voltage-driven net TEA transport were cis-inhibited by other organic cations, and a similar affinity order was found for both transport mechanisms (quinine greater than amiloride greater than morphine greater than mepiperphenidol greater than choline = N1-methylnicotinamide). This data suggests that the same transport protein might be responsible for both phenomena. Other experiments determined that the BLM vesicles had no TEA-H+ exchange, and that contamination of the vesicle population by brush-border membranes was negligible in terms of their contribution to TEA transport. These results demonstrate the presence of an exchange reaction for TEA in the rabbit renal BLM and thus imply carrier-mediated transport of TEA by these membranes.

Acridine Orange↗

PubMed1987

Axial heterogeneity of organic cation transport along the rabbit renal proximal tubule: studies with brush-border membrane vesicles.

Brush-border membrane vesicles prepared from rabbit kidney outer cortex (rich in S1 and S2) and outer medulla (rich in S3) were used to evaluate the axial heterogeneity of tetraethylammonium transport in the proximal tubule. The vesicle preparations had similar Km values but the Vmax values differed, suggesting that axial heterogeneity of tetraethylammonium secretion may be due to differences in transport across the brush-border membrane.

Animals↗