Deletion of the donor splice site of intron 4 in the glucokinase gene causes maturity-onset diabetes of the young.

Missense and nonsense mutations in the glucokinase gene have recently been shown to result in maturity-onset diabetes of the young (MODY), a subtype of non-insulin-dependent diabetes mellitus with early age of onset. Glucokinase catalyzes the formation of glucose-6-phosphate and is involved in the regulation of insulin secretion and integration of hepatic intermediary metabolism. Nucleotide sequence analysis of exon 4 and its flanking intronic regions of the glucokinase gene, in four hyperglycemic individuals of a MODY family, revealed a deletion of 15 base pairs, which removed the t of the gt in the donor splice site of intron 4, and the following 14 base pairs. This deletion resulted in two aberrant transcripts, which were analyzed by reverse transcription of RNA from lymphoblastoid cells obtained from a diabetic patient. In one of the abnormal transcripts, exon 5 is missing, while in the other, the activation of a cryptic splice site leads to the removal of the last eight codons of exon 4. This intronic deletion in a donor splice site seems to cause a more severe form of glucose intolerance, compared with point mutations described in glucokinase. This might be due to a more pronounced effect on insulin secretion.

Impaired hepatic glycogen synthesis in glucokinase-deficient (MODY-2) subjects.

All glucokinase gene mutations identified to date have been localized to exons that are common to the pancreatic and hepatic isoforms of the enzyme. While impaired insulin secretion has been observed in glucokinase-deficient subjects the consequences of this mutation on hepatic glucose metabolism remain unknown. To examine this question hepatic glycogen concentration was measured in seven glucokinase-deficient subjects with normal glycosylated hemoglobin and 12 control subjects using 13C nuclear magnetic spectroscopy during a day in which three isocaloric mixed meals were ingested. The relative fluxes of the direct and indirect pathways of hepatic glycogen synthesis were also assessed using [1-13C]glucose in combination with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. Average fasting hepatic glycogen content was similar in glucokinase-deficient and control subjects (279+/-20 vs 284+/-14 mM; mean+/-SEM), and increased in both groups after the meals with a continuous pattern throughout the day. However, the net increment in hepatic glycogen content after each meal was 30-60% lower in glucokinase-deficient than in the control subjects (breakfast, 46% lower, P < 0.02; lunch, 62% lower, P = 0.002; dinner; 30% lower, P = 0.04). The net increment over basal values 4 h after dinner was 105 +/-18 mM in glucokinase-deficient and 148+/-11 mM in control subjects (P = 0.04). In the 4 h after breakfast, flux through the gluconeogenic pathway relative to the direct pathway of hepatic glycogen synthesis was higher in glucokinase-deficient than in control subjects (50+/-2% vs 34+/-5%; P = 0.038). In conclusion glucokinase-deficient subjects have decreased net accumulation of hepatic glycogen and relatively augmented hepatic gluconeogenesis after meals. These results suggest that in addition to the altered beta cell function, abnormalities in liver glycogen metabolism play an important role in the pathogenesis of hyperglycemia in patients with glucokinase-deficient maturity onset diabetes of young.

Insulin secretory abnormalities in subjects with hyperglycemia due to glucokinase mutations.

Pancreatic beta-cell function was studied in six subjects with mutations in the enzyme glucokinase (GCK) who were found to have elevated fasting and postprandial glucose levels in comparison to six normoglycemic controls. Insulin secretion rates (ISRs) were estimated by deconvolution of peripheral C-peptide values using a two-compartment model and individual C-peptide kinetics obtained after bolus intravenous injections of biosynthetic human C-peptide. First-phase insulin secretory responses to intravenous glucose and insulin secretion rates over a 24-h period on a weight maintenance diet were not different in subjects with GCK mutations and controls. However, the dose-response curve relating glucose and ISR obtained during graded intravenous glucose infusions was shifted to the right in the subjects with GCK mutations and average ISRs over a glucose range between 5 and 9 mM were 61% lower than those in controls. In the controls, the beta cell was most sensitive to an increase in glucose at concentrations between 5.5 and 6.0 mM, whereas in the patients with GCK mutations the point of maximal responsiveness was increased to between 6.5 and 7.5 mM. Even mutations that resulted in mild impairment of in vitro enzyme activity were associated with a > 50% reduction in ISR. The responsiveness of the beta cell to glucose was increased by 45% in the subjects with mutations after a 42-h intravenous glucose infusion at a rate of 4-6 mg/kg per min. During oscillatory glucose infusion with a period of 144 min, profiles from the subjects with mutations revealed reduced spectral power at 144 min for glucose and ISR compared with controls, indicating decreased ability to entrain the beta cell with exogenous glucose. In conclusion, subjects with mutations in GCK demonstrate decreased responsiveness of the beta cell to glucose manifest by a shift in the glucose ISR dose-response curve to the right and reduced ability to entrain the ultradian oscillations of insulin secretion with exogenous glucose. These results support a key role for the enzyme GCK in determining the in vivo glucose/ISR dose-response relationships and define the alterations in beta-cell responsiveness that occur in subjects with GCK mutations.

Clinical phenotypes, insulin secretion, and insulin sensitivity in kindreds with maternally inherited diabetes and deafness due to mitochondrial tRNALeu(UUR) gene mutation.

An A-to-G transition in the mitochondrial tRNALeu(UUR) gene at base pair 3243 has been shown to be associated with the maternally transmitted clinical phenotype of NIDDM and sensorineural hearing loss in white and Japanese pedigrees. We have detected this mutation in 25 of 50 tested members of five white French pedigrees. Affected (mutation-positive) family members presented variable clinical features, ranging from normal glucose tolerance (NGT) to insulin-requiring diabetes. The present report describes the clinical phenotypes of affected members and detailed evaluations of insulin secretion and insulin sensitivity in seven mutation-positive individuals who have a range of glucose tolerance from normal (n = 3) to impaired (n = 1) to NIDDM (n = 3). Insulin secretion was evaluated during two experimental protocols: the first involved the measurement of insulin secretory responses during intravenous glucose tolerance test, hyperglycemic clamp, and intravenous injection of arginine. The second consisted of the administration of graded and oscillatory infusions of glucose and studies to define C-peptide kinetics. This protocol was aimed at assessing two sensitive measures of beta-cell dysfunction: the priming effect of glucose on the glucose-insulin secretion rate (ISR) dose-response curve and the ability of oscillatory glucose infusion to entrain insulin secretory oscillations. Insulin sensitivity was assessed by euglycemic-hyperinsulinemic clamp. Evaluation of insulin secretion demonstrated a large degree of between- and within-subject variability. However, all subjects, including those with NGT, demonstrated abnormal insulin secretion on at least one of the tests. In the four subjects with normal or impaired glucose tolerance, glucose failed to prime the ISR response, entrainment of ultradian insulin secretory oscillations was abnormal, or both defects were present. The response to arginine was always preserved, including in subjects with NIDDM. Insulin resistance was observed only in the subjects with overt diabetes. In conclusion, the pathophysiological mechanisms responsible for the development of NIDDM and insulin-requiring diabetes in this syndrome are complex and might include defects in insulin production, glucose toxicity, and insulin resistance. However, our data suggest that a defect of glucose-regulated insulin secretion is an early possible primary abnormality in carriers of the mutation. This defect might result from the progressive reduction of oxidative phosphorylation and implicate the glucose-sensing mechanism of beta-cells.

Arginine-induced insulin release in glucokinase-deficient subjects.

OBJECTIVE: In eight glucokinase (GCK)-deficient subjects, we have investigated insulin secretion rates (ISRs) in response to intravenous arginine. Impairment in the enzymatic activity of mutant GCK leads to a reduced glycolytic flux in beta-cells. This defect translates in vivo as a right shift in the glucose/SR dose-response curve. Insulin secretion in response to other secretagogues has not been reported. RESEARCH DESIGN AND METHODS: The arginine test was performed immediately after a 2-h hyperglycemic (10 mM) clamp. ISR was computed by deconvolution of peripheral C-peptide levels. Linear regression analyses were performed to assess correlations between the beta-cell secretory responses to the arginine test, an intravenous glucose tolerance test (IVGTT), and a hyperglycemic clamp (areas under the C-peptide curves), and between these parameters and the glucose tolerance status (area under the glucose curve during an oral glucose tolerance test). RESULTS: Two minutes after the injection of arginine, the increment in ISR was 30.17 +/- 10.01 pmol insulin.kg-1.min-1 in patients and 36.25 +/- 15.46 pmol insulin.kg-1.min-1 in control subjects (P = 0.38). Throughout the experiment, increments in ISR were comparable in both groups. The amount of insulin secreted in response to arginine (0-5 min) was similar in patients and control subjects: 81 +/- 28 vs. 119 +/- 55 pmol/kg (P = 0.16), respectively. The arginine test C-peptide response was not correlated with the IVGTT or hyperglycemic clamp responses. The arginine test and hyperglycemic clamp responses were not correlated to the glucose tolerance status. The best predictor of the glucose tolerance was the C-peptide response to the IVGTT (r2 = 0.78; P = 0.002). CONCLUSIONS: beta-cell secretory increment in response to arginine was found to be in the normal range in GCK-deficient subjects. The arginine test does not seem to reflect either the beta-cell secretory defect or the glucose tolerance status of these subjects. IVGTT seems to be the best predictor of the latter parameter in this population.

Molecular plasticity of vascular wall during N(G)-nitro-L-arginine methyl ester-induced hypertension: modulation of proinflammatory signals.

It has previously been reported that hypertension induced by the chronic blockade of NO production is characterized by a proinflammatory phenotype of the arterial wall associated with a periarterial accumulation of inflammatory cells. In the present study, the cellular and molecular mechanisms involved in the luminal and perivascular accumulation of inflammatory cells were evaluated in the aortas of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats. Because the medial layer remains intact, putative markers of the resistance of the vascular wall to cell migration and to oxidative stress were also explored. For this purpose, monocyte adhesion, cytokine expression, superoxide anion production, and nuclear factor-kappa B (NF-kappa B) activation were assessed in the aortas of L-NAME-treated rats. Expressions of tissue inhibitor of metalloproteinases-1 (TIMP-1) and heme oxygenase-1 (HO-1) in the aortic wall were also studied as possible markers of such resistance. Chronic blockade of NO production increased ex vivo monocyte adhesion to the endothelium, increased the production of superoxide anions, and activated the NF-kappa B system. In concert with this modification of the redox state of the vascular wall in L-NAME-treated rats, the expression of proinflammatory cytokines interleukin-6, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor was increased. In parallel, expressions of both TIMP-1 and HO-1 were increased. All these changes were prevented by treatment with an angiotensin-converting enzyme inhibitor (Zofenopril). Hypertension associated with a proinflammatory phenotype of the vascular wall induced by blockade of NO production could be due to an increase in oxidative stress, which, in turn, activates the NF-kappa B system and increases gene expression. In parallel, the arterial wall overexpresses factors such as TIMP-1 and HO-1, which could participate in the resistance to cell migration and oxidative stress.

Guar gum does not impair the absorption and utilization of dietary nitrogen but affects early endogenous urea kinetics in humans.

BACKGROUND: Viscous gums enhance viscosity in the upper gastrointestinal lumen, quickly disturbing motility and promoting fluid secretion. OBJECTIVE: We sought to determine whether guar gum could acutely affect the absorption and utilization of dietary nitrogen and whether these luminal effects could also perturb the kinetics of urea. DESIGN: We studied the short-term effect of adding 1% of highly viscous guar gum to a (15)N-labeled protein meal (30 g soy protein isolate in 500 mL water) during the postprandial phase in humans. The effects on bioavailability were studied by using the [(13)C]glycine breath test (to assess gastric emptying) and (15)N enrichment in plasma amino acids (for systemic amino acid bioavailability). The kinetics of dietary and endogenous urea were assessed in plasma and urine. RESULTS: Guar gum modulated the gastric emptying kinetics of the liquid phase of the meal slightly (P < 0.05), but had no significant effect on either the systemic appearance of dietary amino acids or plasma and urinary dietary urea kinetics. Without significantly affecting plasma urea concentrations, guar gum reduced by approximately 40% the urinary excretion of endogenous urea for the first 2-h period after the meal (P < 0.01), although endogenous urinary excretion was similar at later stages. CONCLUSIONS: Guar gum did not significantly affect the bioavailability or utilization of dietary protein. We showed an early effect of guar gum on endogenous urea kinetics, which most probably arose from very early, short-term stimulation of the intestinal disposal of endogenous urea, at the expense of its urinary excretion.

Angiotensin II-elicited signal transduction via AT1 receptors in endothelial cells.

1. Angiotensin II (AII) actions are mediated by two distinct types of receptors: AT1, which includes two subtypes, AT1A and AT1B, and AT2. AII produces vasoconstriction on the vascular wall acting directly on smooth muscle cells via AT1 receptors. AII receptors have recently been demonstrated on endothelial cells. But the pharmacological characteristics of these receptors and the intracellular signal pathways coupled to them remain unclear. 2. The aim of this work was to characterize the AII receptor subtypes in rat aortic endothelial cells (RAEC) in primary culture and to evaluate the signal pathways coupled to these receptors by measuring the activation of phospholipase C (PLC) and phospholipase A2 (PLA2). 3. Labelled AII bound to RAEC in a specific, saturable manner. Scatchard analysis showed a Kd of 1.87 +/- 0.49 nM and a Bmax of 50.2 +/- 10.9 x 10(3) sites per cell. AII was displaced by the AT1-specific antagonist, DuP753 with a Ki of 17.37 +/- 1.49 nM, but not by the AT2 receptor analogues CGP42771B or PD123177. These data were confirmed by the finding of AT1 mRNA in endothelial cells. Analysis of RNA expression by RT-PCR showed the presence of both subtypes, AT1A and AT1B in endothelial cells, whereas smooth muscle cells express only AT1A. 4. The activation of PLC and PLA2 in response to AII was evaluated by measuring inositol phosphate production and arachidonic acid release, respectively. Both were enhanced by AII in a dose-dependent manner, and inhibited by DuP753, but not by PD123177. 5. We conclude that AT1 receptors are expressed by endothelial cells in primary culture and that phospholipase C and phospholipase A2 activated via this receptor.

Insulin secretion and insulin sensitivity in diabetic and non-diabetic subjects with hepatic nuclear factor-1alpha (maturity-onset diabetes of the young-3) mutations.

OBJECTIVE: To evaluate insulin secretion and sensitivity in affected (diabetes mellitus or impaired glucose tolerance; n=7) and in unaffected (normal glucose tolerance; n=3) carriers of hepatocyte nuclear factor-1alpha (maturity-onset diabetes of the young-3 (MODY3)) gene mutations. METHODS: Insulin secretion was assessed by an i.v. glucose tolerance test (IVGTT), hyperglycemic clamp and arginine test, and insulin sensitivity by an euglycemic hyperinsulinemic clamp. Results were compared with those of diabetic MODY2 (glucokinase-deficient) and control subjects. RESULTS: The amount of insulin secreted during an IVGTT was decreased in affected MODY3 subjects (46+/-24 (s.d.) pmol/kg body weight (BW)) as compared with values in MODY2 (120+/-49pmol/kg BW) and control (173+/-37pmol/kg BW; P=0.0004) subjects. The amount of insulin secreted during a 10mmol/l glucose clamp was decreased in affected MODY3 subjects (171+/-78pmol/kg BW) and MODY2 subjects (302+/-104pmol/kg BW) as compared with control subjects (770+/-199pmol/kg BW; P=0.0001). Insulin secretion in response to arginine was decreased in affected MODY3 subjects. Milder and heterogeneous defects were observed in the unaffected MODY3 subjects; the amount of insulin secreted during the hyperglycemic clamp was 40-79% of that of controls. The response to arginine was abnormally delayed. Insulin sensitivity was decreased in diabetic but not in non-diabetic MODY3 subjects. CONCLUSIONS: Beta-cell dysfunction in response to glucose and arginine is observed in affected and unaffected MODY3 subjects. The MODY3 and MODY2 subtypes present different insulin secretion profiles. Secondary insulin resistance might contribute to the chronic hyperglycemia of MODY3 patients and modulate their glucose tolerance.

Conversion of big-endothelin-1 elicits an endothelin ETA receptor-mediated response in endothelial cells.

Functional conversion of big-endothelin-1 to endothelin-1 and characterization of endothelin receptor subtype were investigated in cultured rat aortic endothelial cells. Exogenous endothelin-1 and big-endothelin-1 both increased arachidonic acid release and inositol phosphate production dose dependently. Endothelin-1 was more potent than big-endothelin-1 as indicated by EC50 values: 0.5 +/- 0.1 nM and 10.0 +/- 2.0 nM for endothelin-1-induced arachidonic acid release and inositol phosphate formation, respectively, versus 1.0 +/- 0.4 nM and 35.0 +/- 6.0 nM for big-endothelin-1-induced responses. Big-endothelin-1, but not endothelin-1 actions were inhibited by phosphoramidon. Comparative studies of endothelin receptor agonists and antagonists showed that endothelin-3 but not sarafotoxin S6c stimulated arachidonic acid release and inositol phosphate formation. The responses to big-endothelin-1 and endothelin-1 were specifically inhibited by the selective endothelin ETA receptor antagonist, [cyclo-D-Trp-D-Asp-Pro-D-Val-Leu] (BQ-123) but not by the selective endothelin ETB receptor antagonist [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma- methyl-Leu-D-Trp-(COMe)-D-NLeu-ONa] (BQ-788). [125I]Endothelin-1 binding was inhibited by endothelin-1, endothelin-3 and BQ-123 but not by BQ-788. These results indicate that the pharmacological responses to big-endothelin-1 in aortic endothelial cells are due to the extracellular phosphoramidon-sensitive conversion to endothelin-1. Endothelin effects are mediated through endothelin ETA receptors in these cells.

A method for obtaining monodispersed cells from isolated porcine islets of Langerhans.

Micro or macroencapsulation of islets of Langerhans have been proposed as a bioartificial pancreas. Encapsulation of dispersed single cells instead of porcine islets should improve the oxygenation of encapsulated tissue. The aim of this work was, therefore, to develop techniques for dissociating porcine islets and test cell viability and function. After islet isolation and purification, islets were dispersed into single cells with collagenase and DNAse in either an extracellular type ionic solution or a UW solution. After culture, islets or cells were perfused with Krebs buffer. Two consecutive stimulations from 2.8 mM to 20 mM glucose were performed. Viability of cells (trypan blue) was higher than 85% after dispersion in ES or UW solutions. Islets or dispersed cells responded similarly to both stimulations with a return to basal rate between stimulations. No difference was found between cell function cultured during 18 hours or 6 days. However, islet function was improved by a long period of culture. In conclusion, this study demonstrates that dissociated cells performed as well as native islets up to six days culture.

Angiotensin II receptors in endothelial cells.

1. Angiotensin II (Ang II), the main effector of the renin-angiotensin system, exerts its vasoconstrictory and trophic actions on smooth muscle cells via AT1 receptors. However, Ang II does not act only on smooth muscle cells, as Ang II receptors are also present in endothelial cells. 2. The receptor type on these cells differs depending on the origin of the endothelium and the species. The rat endothelial receptors are mostly of the AT1 type, but AT2 receptors have also been found. The pharmacological characteristics of the AT1 receptors on endothelial cells are similar to those of other cell types. 3. Ang II stimulates phospholipase C and phospholipase A2 activation via the AT1 receptor in endothelial cells. Ang II also stimulates the tyrosine phosphorylation of several proteins in these cells. 4. Some studies suggest that the AT1 receptor mediates the release of vasodilator molecules by endothelial cells and could modulate Ang II effect on smooth muscle cells. Ang II may also inhibit endothelial cell growth via the AT2 receptor. Finally, endothelial Ang II receptors may be implicated in the regulation of fibrinolysis.

In vitro activation of human macrophages by alginate-polylysine microcapsules.

Microencapsulated islets of Langerhans have been proposed as a bioartificial pancreas. However, foreign body reaction with fibrosis has been observed around implanted microcapsules. Since macrophages are present in this reaction and interleukin-1 (IL-1), a cytokine released by activated macrophages, may induce fibrosis, we tested the capacity of alginate-polylysine microcapsules to activate macrophages. Human monocytes were isolated from whole blood of healthy donors by a Ficoll density gradient and adherence to a plastic support. Monocytes were cultured for 24 h with: (1) alginate-polylysine microcapsules; (2) lipopolysaccharide (LPS) (positive control group); and (3) alone (negative control group). Monocyte activation was evaluated by measuring the secretion of IL-1 beta and the production of intracellular IL-1 alpha and IL-1 beta. Macrophages characterization was performed by immunocytological subtyping. IL-1 beta release and intracellular IL-1 beta and IL-1 alpha production were significantly higher when macrophages were cultured with alginate-polylysine microcapsules than when macrophages were cultured alone. In conclusion, macrophages are activated in vitro by alginate-polylysine microcapsules. This effect may be involved in the fibrosis observed in vivo around implanted microcapsules. In addition, interleukin-1, released during macrophage activation, may cross the microcapsule membrane and impair islet function.

Complement activation by alginate-polylysine microcapsules used for islet transplantation.

A foreign body reaction is frequently observed around implanted microcapsules of alginate-polylysine. Since complement activation can play a role in this reaction, we checked in vitro the ability of empty alginate-polylysine microcapsules to activate complement. Human serum was incubated with microcapsules, and complement activation was evaluated by two methods: the complement hemolytic activity (CH50) and the assay of the C3adesArg fragment. The occurrence of complement activation in the presence of microcapsules was suggested both by a CH50 decrease and by high C3adesArg levels despite C3adesArg adsorption to the capsule membrane. Capsule membrane protection against the cytotoxic effects of complement was also tested. No hemolysis occurred when microencapsulated sensitized sheep erythrocytes were incubated with activated complement. In conclusion, the microcapsule membrane can protect cells against activated complement fragments. Nevertheless, alginate-polylysine microcapsules do activate complement, and this effect must be considered for its use as an implant.

Insulin secretion in rats with chronic nitric oxide synthase blockade.

Nitric oxide, which is produced from L-ar-ginine by a nitric oxide-synthase enzyme, has been shown to be a ubiquitous messenger molecule. Recently, it has been suggested that nitric oxide might influence insulin secretion by activating the soluble guanylate cyclase and generating cyclic guanosine monophosphate (cGMP). We have investigated the role of the nitric oxide pathway in insulin secretion by evaluating the insulin response to several secretagogues in rats in which nitric oxide-synthase was chronically inhibited by oral administration of the L-arginine analogue, NG-nitro-L-arginine methyl ester (L-NAME). Blood pressure and aortic wall cGMP content were used as indices of nitric oxide-synthase blockade. Insulin secretion was evaluated after an intravenous bolus of D-glucose, L-arginine or D-arginine. Chronic L-NAME administration induced a 30% increase in blood pressure and a seven-fold drop in arterial cGMP content. Body weight, fasting plasma glucose and insulin were not influenced by L-NAME administration. First-phase insulin secretion (1 + 3 min) in response to glucose was not significantly different in L-NAME and control rats. The areas under the insulin curve were similar in both groups. Insulin secretion in response to D-arginine or L-arginine in L-NAME-treated and control rats were also similar. In conclusion, chronic nitric oxide-synthase blockade increases blood pressure and decreases aortic cGMP content, but does not alter insulin secretion in response to several secretagogues. Chronic oral administration of L-NAME in the rat provides an adequate animal model for studying the L-arginine nitric oxide-pathway.

Differential expression of rat frizzled-related frzb-1 and frizzled receptor fz1 and fz2 genes in the rat aorta after balloon injury.

Frzb-1 is a secreted protein, presenting similarity with the Wnt-binding domain of the frizzled family of receptors, which acts as an antagonist of Wnt signaling. Using mRNA differential display in the rat aorta balloon injury model, we identified overexpression of Frzb-1 mRNA and determined its cDNA sequence. By quantitative reverse transcription-polymerase chain reaction and RNase protection assay, a biphasic upregulation of rFrzb-1 expression was observed, with significant peaks of a 1.7-fold increase at 4 days and a 1. 5-fold increase at 3 weeks after aortic injury in vivo. In contrast, expression of the rat frizzled receptor genes rfz1 and rfz2 were transiently downregulated at 1 and 4 hours after balloon injury. rFrzb-1 was expressed predominantly in rat aortic smooth muscle cells (RASMCs) and barely in aortic fibroblasts and endothelial cells (RAECs), whereas rfz1 and rfz2 were expressed in all of these cells when stimulated with serum. Transient downregulation of rfz1 and rfz2 expression was reproduced by stimulation of quiescent RASMCs with serum, platelet-derived growth factor-BB, or fibroblast growth factor-2. In contrast, rFrzb-1 expression diminished slowly, to reach a 2-fold decrease 24 hours after growth factor stimulation, implying that quiescent RASMCs expressed higher levels of rFrzb-1 mRNA than did proliferative ones. Overexpression of rFrzb-1 in the aorta seemed to coincide with the arrest of RASMC proliferation occurring in the media 4 days and in the neointima 3 weeks after balloon injury. Our results demonstrate that rfrzb-1, rfz1, and rfz2 are differentially regulated in response to arterial injury and that this modulation seems to follow the proliferative state of RASMCs, suggesting that these Wnt-signaling components may be involved in intimal vascular disease.

Regulation of vascular endothelial growth factor expression by cAMP in rat aortic smooth muscle cells.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.

Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells.

Angiotensin II (ANG II) produces vasoconstriction by a direct action on smooth muscle cells via AT1 receptors. These receptors are also present in the endothelium, but their function is poorly understood. This study was therefore undertaken to determine whether ANG II elicits the release of nitric oxide (NO) from cultured rat aortic endothelial cells. NO production, measured by the accumulation of nitrite and nitrate, was enhanced by 10(-7) M ANG II. The biological activity of the NO released by ANG II action was evaluated by measuring its guanylate cyclase-stimulating activity in smooth muscle cells. The guanosine 3',5'-cyclic monophosphate (cGMP) content of smooth muscle cells was significantly increased by exposure of supernatant from ANG II-stimulated endothelial cells. These effects resulted from the activation of NO synthase, as they were inhibited by the L-arginine analogs. These ANG II actions were mediated by the AT1 receptor, as shown by their inhibition by the AT1 antagonist losartan. The cGMP production by reporter cells was inhibited by the calmodulin antagonist W-7, suggesting that ANG II activates endothelial calmodulin-dependent NO synthase. This hypothesis is also supported by the increase of intracellular free calcium induced by ANG II in endothelial cells. ANG II also stimulated luminol-enhanced chemiluminescence in endothelial cells. This effect was inhibited by N omega-monomethyl-L-arginine and superoxide dismutase, suggesting that this luminol-enhanced chemiluminescence reflected an increase in peroxynitrite production. Thus ANG II stimulates NO release from macrovascular endothelium, which may modulate the direct vasoconstrictor effect of ANG II on smooth muscle cells. However, this beneficial effect may be counteracted by the simultaneous production of peroxynitrite, which could contribute to several pathological processes in the vascular wall.

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells.

The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.

The influence of the albumin fraction on the bioavailability and postprandial utilization of pea protein given selectively to humans.

Pulse seed proteins such as those found in peas (Pisum sativum) contain fractions of very dissimilar composition and properties, which may therefore be differently utilized by the human body. To analyze the nutritional value of the soluble protein fractions of pea seed, human volunteers ingested a mixed meal of 30 g of raw purified pea protein either as [15N]-globulins (G, n = 9) or as a mix of [15N]-globulins and [15N]-albumins (GA, n = 7) in their natural proportions (22:8). Dietary and endogenous nitrogen fluxes at the terminal ileum were assessed using a tube perfusion technique with an isotopic dilution method. Systemic dietary amino acid availability and the retention of dietary amino acids were determined using 15N enrichment in plasma amino acids and deamination products in blood and urine for 8 h postprandially. The results showed that the pea albumin fraction had the following effects: 1) significantly lowered the real ileal digestibility of pea protein (94 +/- 2.5% for G vs. 89.9 +/- 4% for GA), probably because of a direct effect of trypsin inhibitors; 2) did not promote acute intestinal losses of endogenous nitrogen; and 3) did not significantly improve the postprandial biological value of pea protein (76.5 +/- 3.9% for G vs. 78.7 +/- 3.6% for GA), despite the fact that it corrected the globulin deficiency in sulfur amino acids. We conclude that both G and GA are of good nutritional value for humans and show that cysteine-rich albumins have a far more modest effect on the efficiency of postprandial dietary protein utilization than would be expected from the amino acid scores.