Nitric oxide generation mediated by β-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Aims/hypothesis Type 2 diabetic patients have been shown to have reduced basal platelet nitric oxide synthase activity, which is a possible contributor to the vascular complications seen in the disease. We investigated platelet nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase in Type 2 diabetic patients and control subjects.Methods Platelets isolated from blood taken from nine Type 2 diabetic patients and nine healthy control subjects of similar age were treated with isoproterenol 1 µmol/l, forskolin 1 µmol/l or vehicle. Platelet nitric oxide synthase activity was measured by L-[³H]-arginine to L-[³H]-citrulline conversion, cyclic GMP content by radioimmunoassay, and nitric oxide synthase type 3 expression by western blotting.Results Basal platelet nitric oxide synthase activity was lower in diabetic patients than in control subjects (0.01±0.02 pmol L-citrulline/10⁸ platelets, compared with 0.12±0.05; p<0.05), although no corresponding difference was seen in basal platelet cyclic GMP (0.61±0.39 and 0.13±0.22 pmol cyclic GMP/10⁸ platelets respectively; p=0.37). In control subjects isoproterenol 1 µmol/l and forskolin 1 µmol/l increased platelet nitric oxide synthase activity (to 0.27±0.08 and 0.27±0.07 pmol L-citrulline/10⁸ platelets respectively; p<0.05 for each in comparison with basal) and cyclic GMP (to 1.84±0.41 and 1.86±0.48; p<0.05 for each in comparison with basal). This effect was not achieved in diabetic patients. Isoproterenol- and forskolin-stimulated cyclic GMP correlated inversely with plasma glucose and HbA_1c. Platelet nitric oxide synthase type 3 expression was not different in control and diabetic subjects and was not changed by acute exposure of platelets to isoproterenol.Conclusions/interpretation Nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase is impaired in platelets of people with Type 2 diabetes mellitus, with no corresponding change in nitric oxide synthase type 3 expression. It is possible that this impairment contributes to the thrombotic and atherosclerotic complications of Type 2 diabetes.Abbreviations AR -adrenoceptors - GFP gel-filtered platelets - L-NAME N^G-nitro-L-arginine methyl ester - L-NMMA N^G-monomethyl-L-arginine - NO nitric oxide - NOS2 nitric oxide synthase type 2 - NOS3 nitric oxide synthase type 3     

Negative effect of nitric oxide on shorteningfrequency relationship in cardiac myocytes is diminished after simulated ischemia-reperfusion

Increasing stimulation rate increases function in cardiac myocytes and nitric oxide and cyclic GMP inhibit this effect. We tested the hypothesis that myocyte stunning would blunt both the effects of increases in rate and of nitric oxide and cyclic GMP. Ventricular myocytes from 11 rabbits were used to determine maximum rate of shortening (R_max, µm/s) and %shortening during control and after simulated ischemia [15 min 95% N₂- 5% CO₂] and reperfusion [reoxygenation]. Measurements were obtained at 1–4 Hz with vehicle, 1H[1,2,4]oxadiazolo[4,3,alpha] quinoxaline-1-one (ODQ) 10^–6 M, soluble guanylyl cyclase inhibitor, or N^G-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor (L-NAME) 10^–5 M. In control, increases in rate increased R_max from 69 ± 3 to 254±12 and %shortening from 5.3 ± 0.3 to 8.7 ± 0.5. Both ODQ and L-NAME shifted values higher. With stunning, the effects of pacing on Rmax and %shortening were blunted and ODQ and L-NAME failed to alter these values. Cyclic GMP was 322±37 pmol/10⁵ myocytes at baseline and these values were lowered by ODQ (244 ± 31) and LNAME (207 ± 23), and similar changes were observed in stunned myocytes. Increasing frequency increased function, and reducing nitric oxide/cyclic GMP enhanced this relationship. The effect of nitric oxide was diminished by stunning, but this was not related to altered cyclic GMP levels. This suggested changes in effects of cyclic GMP downstream to its production during myocardial stunning.     

Interleukin-1β effects on cyclic GMP and cyclic AMP in cultured rat islets of Langerhans — arginine — dependence and relationship to insulin secretion

Summary When islets were cultured with interleukin-1 (1 or 100 pmol/l) for 12 h in arginine-containing medium, cyclic GMP levels were increased 1.6- and 4.5-fold respectively. The arginine analogue, N--nitro-l-arginine methyl ester, which blocks nitric oxide formation and partially reverses inhibition of insulin secretion by 100 pmol/l interleukin-1, largely, but not completely, blocked generation of cyclic GMP. Treatment of islets with 100 pmol/l interleukin-1 for 12 h significantly decreased islet cyclic AMP generation in the absence of isobutylmethylxanthine (from 13.1±0.7 to 9.3±0.8 fmol/g islet protein), this fall was arginine-dependent and may have resulted from an effect on a cyclic AMP phosphodiesterase, since it was masked if isobutylmethylxanthine was present. Isobutylmethylxanthine (0.4 mmol/l) reduced the inhibitory potency of interleukin-1 in 15 h slightly but significantly from 80.5 to 59.0%. The morpholinosydnonimine SIN-1, which is a nitric oxide donor, inhibited insulin secretion, raised islet cyclic GMP and lowered cyclic AMP; its effects were similar to those of interleukin-1. However, 6-anilinoquinoline-5,8-quinone, [LY83583 (1–10 mol/l)], inhibited insulin secretion, and significantly decreased cyclic GMP while 8-bromocyclic GMP stimulated insulin secretion. Both low- and high-dose interleukin-1 treatment give a large arginine-dependent and a small, yet significant, arginine-independent increase in cyclic GMP. The inhibitory effect of SIN-1 or interleukin-1 on insulin secretion seems to depend to a small extent on decreased islet cyclic AMP, though sustained increases in nitric oxide or depleted islet GTP may directly affect the secretory process.     

High affinity binding sites for proinsulin in human IM-9 lymphoblasts

Summary Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity of receptors, or both. Using flow-cytometric analysis of insulin binding, we were able to distinguish and separate two different IM-9 cell fractions. In both fractions, Scatchard plots for specific binding of insulin and proinsulin were linear, suggesting the presence of two distinct populations of receptors. Type 1 cells showed low capacity but high affinity of insulin binding (16,300±3,000 sites/cell; K_d 0.4±0.1 nmol/l). Proinsulin and insulin-like growth factor 1 (IGF-1) were significantly less potent in competition. MA-20, a specific antibody against human insulin receptors, inhibited insulin binding by 80%, while the specific antibody against human IGF-1 receptors, IR-3, had no effect. Pretreatment with insulin decreased insulin binding by 90%. ¹²⁵I-insulin displayed stepwise dissociation with the rate markedly enhanced by cold insulin. Type 2 cells exhibited significantly different binding characteristics with higher capacity but lower affinity of ¹²⁵I-insulin binding (430,000±25,000 sites/cell, _p<0.001 vs type 1; K_d 2±0.4 nmol/l, p<0.02 vs type 1). Proinsulin competed with similar potency for insulin binding, while IGF-1 was still less potent. ¹²⁵I-proinsulin showed a significantly higher binding affinity than ¹²⁵I-insulin (K_d 0.5±0.3 nmol/l, p<0.05) with 50,000±10,000 binding sites/cell. C-peptide was able to compete for ¹²⁵I-proinsulin, but not for ¹²⁵I-insulin binding. MA-20 did not influence ¹²⁵I-proinsulin binding, but inhibited ¹²⁵I-insulin binding by 50%, whereas IR-3 increased proinsulin binding 1.5-fold with no effect on insulin binding. Preincubation with insulin decreased insulin binding by 50% and proinsulin binding by 10%. The dissociation of ¹²⁵I-proinsulin showed linear first-order kinetics and was not significantly accelerated by cold proinsulin. Furthermore, the tyrosine phosphorylation of a 65 kDa protein was stimulated to a significantly greater extent by proinsulin than by insulin, indicating activation of different signalling cascades. DNA analysis revealed that type 1 cells were predominantly in the G₁ phase, whereas type 2 cells were in the S and G₂ + M phases of the cell cycle. We conclude, that IM-9 lymphoblasts were separated by flow-cytometry into one fraction with typical insulin receptors and a second fraction with high affinity binding sites for proinsulin. High affinity proinsulin binding sites were distinguished from typical insulin receptors by: 1) higher affinity for proinsulin than insulin, 2) inhibition of proinsulin binding by C-peptide but not by the insulin receptor antibody MA-20, 3) non-co-operative first order dissociation kinetics of proinsulin binding, 4) resistance to down-regulation by insulin, and 5) differences in signal transduction.Abbreviations IR-3 Monoclonal anti-IGF-1 receptor antibody - BSA bovine serum albumin - FACS fluorescence-activated cell sorting - FCS fetal calf serum - FITC fluorescein isothiocyanate - G₁, S, G₂ + M cell cycle phases - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] - IGF-1/2 insulin-like growth factor-1/2 - MA-20 monoclonal anti-insulin receptor antibody - NIDDM non-insulin-dependent diabetes mellitus - PBS phosphate buffered saline - TCA trichloroacetic acid - type 1 IM-9 cell fraction with low insulin binding capacity - type 2 IM-9 cell fraction with high insulin binding capacity     

Distribution of Nitric Oxide Synthase and Secretory Role of Exogenous Nitric Oxide in the Isolated Rat Pancreas

Summary Background. Pancreatic production and in vivo effects of nitric oxide (NO) have been shown by several studies. In order to examine the direct actions of the NO donor sodium nitroprusside (SNP), this study used in vitro specimens of the rat pancreas where the distribution of neuronal nitric oxide synthase (NOS) and the secretory effects of SNP and the cyclic GMP (cGMP) analog 8-bromo cyclic GMP (8-Br cGMP) were investigated. Methods. NO containing pancreatic nerves were visualized by NOS immunohistochemistry. Basal and stimulated amylase output from rat pancreatic segments was measured by an on-line fluorimetric method Stimulation was achieved by either acetylcholine (ACh) or electrical field stimulation (EFS). Intracellular free calcium concentration ([Ca²⁺]_i) was measured in dispersed pancreatic acinar cells. Results. NOS containing nerves were demonstrated in the vicinity of pancreatic acini and blood vessels. SNP and 8-Br cGMP inhibited both basal and EFS evoked amylase output but failed to inhibit ACh induced amylase output. Basal [Ca²⁺]_i was decreased by both SNP and 8-Br cGMP but neither SNP nor 8-Br cGMP influenced the ACh evoked increase in [Ca²⁺]_i. Conclusion. NO is well distributed in the rat exocrine pancreas. Exogenous nitric oxide may have a dual action in the isolated rat pancreas: Inhibition of basal amylase secretion in acinar cells and inhibition of ACh release from intrinsic nere terminals. Both effects seem to be calcium dependent and possibly mediated by cGMP.     

Methylglyoxal impairs endothelial insulin sensitivity both in vitro and in vivo

Aims/hypothesis

Insulin exerts a direct action on vascular cells, thereby affecting the outcome and progression of diabetic vascular complications. However, the mechanism through which insulin signalling is impaired in the endothelium of diabetic individuals remains unclear. In this work, we have evaluated the role of the AGE precursor methylglyoxal (MGO) in generating endothelial insulin resistance both in cells and in animal models.

Methods

Time course experiments were performed on mouse aortic endothelial cells (MAECs) incubated with 500 μmol/l MGO. The glyoxalase-1 inhibitor S-p-bromobenzylglutathione-cyclopentyl-diester (SpBrBzGSHCp2) was used to increase the endogenous levels of MGO. For the in vivo study, an MGO solution was administrated i.p. to C57BL/6 mice for 7 weeks.

Results

MGO prevented the insulin-dependent activation of the IRS1/protein kinase Akt/endothelial nitric oxide synthase (eNOS) pathway, thereby blunting nitric oxide (NO) production, while extracellular signal-regulated kinase (ERK1/2) activation and endothelin-1 (ET-1) release were increased by MGO in MAECs. Similar results were obtained in MAECs treated with SpBrBzGSHCp2. In MGO- and SpBrBzGSHCp2-exposed cells, inhibition of ERK1/2 decreased IRS1 phosphorylation on S616 and rescued insulin-dependent Akt activation and NO generation, indicating that MGO inhibition of the IRS1/Akt/eNOS pathway is mediated, at least in part, by ERK1/2. Chronic administration of MGO to C57BL/6 mice impaired whole-body insulin sensitivity and induced endothelial insulin resistance.

Conclusions/interpretation

MGO impairs the action of insulin on the endothelium both in vitro and in vivo, at least in part through an ERK1/2-mediated mechanism. These findings may be instrumental in developing novel strategies for preserving endothelial function in diabetes.     

Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect

Summary Insulin (100 U/l) stimulated xylose uptake in rat soleus muscle from a basal value of 2.3±0.5 to 11.6±2.1 mol · g^-1 · h^-1. Denervation (section of the sciatic nerve) markedly reduced the stimulatory action of insulin (basal 1.3 ±0.4 mol · g^-1 · h^-1; insulin-stimulated 4.5±0.6 mol · g^-1 · h^-1). This effect appeared 3 days after denervation and was maximal after 5 days. Denervation also affected the insulin dose response curve; there was no effect of insulin in denervated muscle until the concentration exceeded 1 U/l. Denervation inhibited insulin-stimulated -aminoisobutyrate uptake by 77% but did not affect ¹²⁵I-insulin binding or glucose-independent activation of glycogen synthase by insulin. There was no effect of denervation on the insulinomimetic effects of concanavalin A, hydrogen peroxide, vitamin K₅, anoxia, 24-dinitrophenol, cooling, hyperosmolarity or EDTA, but the effect of diamide was inhibited. It is concluded [1] that denervation inhibits insulin-stimulated sugar transport at some early post-receptor step, and [2] that the mechanism whereby insulin activates glycogen synthase is different from the activation of the membrane transport of sugars and amino acids.     

Cyclic nucleotide phosphodiesterases in pancreatic islets

Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of enzymes (PDE1-PDE11) which hydrolyse cyclic AMP and cyclic GMP to their biologically inactive 5 derivatives. Cyclic AMP is an important physiological amplifier of glucose-induced insulin secretion. As PDEs are the only known mechanism for inactivating cyclic nucleotides, it is important to characterise the PDEs present in the pancreatic islet beta cells. Several studies have shown pancreatic islets or beta cells to contain PDE1C, PDE3B and PDE4, with some evidence for PDE10A. Most evidence suggests that PDE3B is the most important in relation to the regulation of insulin release, although PDE1C could have a role. PDE3-selective inhibitors augment glucose-induced insulin secretion. In contrast, activation of beta-cell PDE3B could mediate the inhibitory effect of IGF-1 and leptin on insulin secretion. In vivo, although PDE3 inhibitors augment glucose-induced insulin secretion, concomitant inhibition of PDE3B in liver and adipose tissue induce insulin resistance and PDE3 inhibitors do not induce hypoglycaemia. The development of PDE3 inhibitors as anti-diabetic agents would require differentiation between PDE3B in the beta cell and that in hepatocytes and adipocytes. Through their effects in regulating beta-cell cyclic nucleotide concentrations, PDEs could modulate beta-cell growth, differentiation and survival; some work has shown that selective inhibition of PDE4 prevents diabetes in NOD mice and that selective PDE3 inhibition blocks cytokine-induced nitric oxide production in islet cells. Further work is required to understand the mechanism of regulation and role of the various PDEs in islet-cell function and to validate them as targets for drugs to treat and prevent diabetes.Abbreviations PDE phosphodiesterase - GIP glucose-dependent insulinotropic peptide - GLP-1 glucagon like peptide 1 - PDX-1 pancreatic duodenal homeobox-1 - IBMX isobutylmethylxanthine     

Endothelial control of coronary flow in perfused guinea pig heart

In perfused isolated guinea pig hearts reactive hyperemia (RH) was induced by occlusion of coronary flow for periods ranging from 1–60 s. RH was hampered by 100–60% in the presence of an inhibitor of NO synthase, N^G-nitro-L-arginine (100 M) and, to a lesser extent (up to 35%), by an antagonist of adenosine receptors, 8-phenyltheophylline (10 M). An inhibitor of PGH synthase, indomethacin (5 M), did not affect RH. During RH the heart generated prostacyclin, nitric oxide, and adenosine as indicated by the appearance of 6-keto-PGF₁, cyclic GMP, urate, inosine, hypoxanthine and xanthine in the perfusate. Out of these factors only NO and adenosine were responsible for RH. NO was responsible for RH which was evoked by short-term (1–10 s) coronary occlusion, whereas concurrent efforts of NO and adenosine were required to maintain RH that followed longer (20–60 s) periods of interruption of coronary inflow. Thus, in the investigated system nitric oxide and adenosine but not prostacyclin can be considered as the mediators of myocardial reactive hyperemia.Invited Contributions to the Symposium Regulation of coronary blood flow, held at the XV. World Congress of the International Society for Heart Research in Prague 1995     

Glucose effectiveness,but not insulin sensitivity,is improved after short-term interval training in individuals with type 2 diabetes mellitus: a controlled,randomised, crossover trial

Aims/hypothesis

The role of glucose effectiveness (S _G) in training-induced improvements in glucose metabolism in individuals with type 2 diabetes is unknown. The objectives and primary outcomes of this study were: (1) to assess the efficacy of interval walking training (IWT) and continuous walking training (CWT) on S _G and insulin sensitivity (S _I) in individuals with type 2 diabetes; and (2) to assess the association of changes in S _G and S _I with changes in glycaemic control.

Methods

Fourteen participants with type 2 diabetes underwent three trials (IWT, CWT and no training) in a crossover study. Exclusion criteria were exogenous insulin treatment, smoking, pregnancy, contraindications to structured physical activity and participation in recurrent training (>90 min/week). The trials were performed in a randomised order (computerised-generated randomisation). IWT and CWT consisted of ten supervised treadmill walking sessions, each lasting 60 min, over 2 weeks. IWT was performed as repeated cycles of 3 min slow walking and 3 min fast walking (aiming for 54% and 89% of \( \overset{\cdotp }{V}{\mathrm{O}}_{2\mathrm{peak}} \), respectively, which was measured during the last minute of each interval), and CWT was performed aiming for a moderate walking speed (73% of \( \overset{\cdot }{V}{\mathrm{O}}_{2\mathrm{peak}} \)). A two-step (pancreatic and hyperinsulinaemic) hyperglycaemic clamp was implemented before and after each trial. All data were collected in a hospitalised setting. Neither participants nor assessors were blinded to the trial interventions.

Results

Thirteen individuals completed all procedures and were included in the analyses. IWT improved S _G (mean ± SEM: 0.6 ± 0.1 mg kg^?1 min^?1, p < 0.05) but not S _I (p > 0.05), whereas CWT matched for energy expenditure and time duration improved neither S _G nor S _I (both p > 0.05). Changes in S _G, but not in S _I, were associated with changes in mean (β = ?0.62 ± 0.23, r ² = 0.17, p < 0.01) and maximum (β = ?1.18 ± 0.52, r ² = 0.12, p < 0.05) glucose levels during 24 h continuous glucose monitoring.

Conclusions/interpretation

Two weeks of IWT, but not CWT, improves S _G but not S _I in individuals with type 2 diabetes. Moreover, changes in S _G are associated with changes in glycaemic control. Therefore, increased S _G is likely an important mechanism by which training improves glycaemic control in individuals with type 2 diabetes.

Trial registration:

ClinicalTrials.gov NCT02320526

Funding:

CFAS is supported by a grant from TrygFonden. During the study period, the Centre of Inflammation and Metabolism (CIM) was supported by a grant from the Danish National Research Foundation (DNRF55). The study was further supported by grants from Diabetesforeningen, Augustinusfonden and Krista og Viggo Petersens Fond. CIM/CFAS is a member of DD2—the Danish Center for Strategic Research in Type 2 Diabetes (the Danish Council for Strategic Research, grant no. 09–067009 and 09–075724).

     

β<Subscript>2</Subscript>-Adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signalling pathway independent of cyclic AMP

Aims/hypothesis

In skeletal muscle, the storage of glycogen by insulin is regulated by glycogen synthase, which is regulated by glycogen synthase kinase 3 (GSK3). Here we examined whether adrenergic receptor activation, which can increase glucose uptake, regulates glycogen synthesis in L6 skeletal muscle cells.

Methods

We used L6 cells and measured glycogen synthesis (as incorporation of d-[U-¹⁴C]glucose into glycogen) and GSK3 phosphorylation following adrenergic activation.

Results

Insulin (negative logarithm of median effective concentration [pEC₅₀] 8.2?±?0.3) and the β-adrenergic agonist isoprenaline (pEC₅₀ 7.5?±?0.3) induced a twofold increase in glycogen synthesis in a concentration-dependent manner. The α₁-adrenergic agonist cirazoline and α₂-adrenergic agonist clonidine had no effect. Both insulin and isoprenaline phosphorylated GSK3. The β-adrenergic effect on glycogen synthesis is mediated by β₂-adrenoceptors and not β₁-/β₃-adrenoceptors, and was not mimicked by 8-bromo-cyclic AMP or cholera toxin, and also was insensitive to pertussis toxin, indicating no involvement of cyclic AMP or inhibitory G-protein (G_i) signalling in the β₂-adrenergic effect on glycogen synthesis. 12-O-tetra-decanoylphorbol-13-acetate (TPA) increased glycogen synthesis 2.5-fold and phosphorylated GSK3 fourfold. Inhibition of protein kinase C (PKC) isoforms with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrollo(3,4-c)-carbazole (Gö6976; inhibits conventional and novel PKCs) or 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983; inhibits conventional, novel and atypical PKCs) inhibited the stimulatory TPA effect, but did not significantly inhibit glycogen synthesis mediated by insulin or isoprenaline. Inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin inhibited the effects of insulin and isoprenaline on glycogen synthesis.

Conclusions/interpretation

These results demonstrate that in L6 skeletal muscle cells adrenergic stimulation through β₂-adrenoceptors, but not involving cyclic AMP or G_i, activates a PI3K pathway that stimulates glycogen synthesis through GSK3.

     

Dehydroepiandrosterone stimulates endothelial proliferation and angiogenesis through extracellular signal-regulated kinase 1/2-mediated mechanisms

Dehydroepiandrosterone (DHEA) activates a plasma membrane receptor on vascular endothelial cells and phosphorylates ERK 1/2. We hypothesize that ERK1/2-dependent vascular endothelial proliferation underlies part of the beneficial vascular effect of DHEA. DHEA (0.1-10 nm) activated ERK1/2 in bovine aortic endothelial cells (BAECs) by 15 min, causing nuclear translocation of phosphorylated ERK1/2 and phosphorylation of nuclear p90 ribosomal S6 kinase. ERK1/2 phosphorylation was dependent on plasma membrane-initiated activation of Gi/o proteins and the upstream MAPK kinase because the effect was seen with albumin-conjugated DHEA and was blocked by pertussis toxin or PD098059. A 15-min incubation of BAECs with 1 nm DHEA (or albumin-conjugated DHEA) increased endothelial proliferation by 30% at 24 h. This effect was not altered by inhibition of estrogen or androgen receptors or nitric oxide production. There was a similar effect of DHEA to increase endothelial migration. DHEA also increased the formation of primitive capillary tubes of BAECs in vitro in solubilized basement membrane. These rapid DHEA-induced effects were reversed by the inhibition of either Gi/o-proteins or ERK1/2. Additionally, DHEA enhanced angiogenesis in vivo in a chick embryo chorioallantoic membrane assay. These findings indicate that exposure to DHEA, at concentrations found in human blood, causes vascular endothelial proliferation by a plasma membrane-initiated activity that is Gi/o and ERK1/2 dependent. These data, along with previous findings, define an important vascular endothelial cell signaling pathway that is activated by DHEA and suggest that this steroid may play a role in vascular function.     

Insulin secretion in rats with chronic nitric oxide synthase blockade

Summary Nitric oxide, which is produced from l-arginine 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, N^G-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.Abbreviations NO Nitric oxide - cGMP guanosine 3: 5 cyclic monophosphate - l-NMMA N^G-monomethyl-l-arginine - l-NAME N^G-nitro-l-arginine-methyl-ester - NOD mice non obese diabetic mice     

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Summary Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. ¹²⁵I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro³⁴]-NPY > NPY 3–36 > > NPY 13–36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits ¹²⁵I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (G_i or G_o) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7–36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through G_i to the inhibition of adenylyl cyclase. [Diabetologia (1998) 41: 1482–1491] Received: 10 November 1997 and in final revised form: 16 June 1998     

Adrenergic receptors coupled to adenylate cyclase in human cerebromicrovascular endothelium

Cultured endothelium derived from three microvascular fractions of human brain was used to characterize adrenergic receptors coupled to adenylate cyclase activity. Catecholamines (norepinephrine, epinephrine) and their analogs (isoproterenol, phenylephrine, 6-fluoronorepinephrine) dose-dependently stimulated endothelial production of cAMP. Antagonists for ß₁ and ß₂receptors (propranolol, atenolol, and butoxamine) and for 1-receptors (prazosin) dose-dependently blocked cAMP formation induced by the tested adrenergic agonists. Clonidine, an ga₂>₁-agonist, also inhibited isoproterenol-stimulated production of cAMP while yohimbine (₂>₁ antagonist) augmented the norepinephrine or epinephrine-induced accumulation of cAMP. Cholera toxin-induced ADP ribosylation of the stimulatory guanine nucleotide binding protein (G_s) abolished the stimulatory effect of norepinephrine, epinephrine, phenylephrine or 6-fluoronorepinephrine on cAMP formation. ADP ribosylation of the inhibitory guanine nucleotide binding protein (G_i) by pertussis toxin had no effect on either phenylephrine-or 6-fluoronorepinephrine-induced production of cAMP while it increased the norepinephrine and epinephrine-induced accumulation of cAMP. These findings represent the first documentation of ß₁-, ß₂-, ₁ and 2-adrenergic receptors linked to adenylate cyclase in endothelium derived from human brain microvasculature. These data also indicate that activation of endothelial ₁ -adrenergic receptors is mediated by a signal transduction mechanism associated with G_s protein. The results strongly support the presence of various receptor-controlled adrenergic regulatory mechanisms on human cerebromicrovascular endothelium.     

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone

Summary It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240–960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7±0.1 to 2.6±0.4 pmol/10⁶ cells, p=0.0001; cGMP: from 1.3±0.2 to 3.4±0.7 pmol/10⁶ cells, p=0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p=0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor N^G-monomethyl-l-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p=0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p=0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.Abbreviations cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - PDE phosphodiesterases - NO nitric oxide - hVSMC human vascular smooth muscle cells - l-NMMA N^G-monomethyl-l-arginine - GTN glyceryltrinitrate - BSA bovine serum albumin - NIDDM non-insulin-dependent diabetes mellitus - MEM minimal essential medium - RIA radioimmunoassay     

17β-Estradiol Exposure Leads to Activation of ERK and p38 but Not JNK in Vascular Endothelial Cells

Estrogen has been shown to increase endothelial cell (EC) production of nitric oxide (NO) by the rapid activation of endothelial nitric oxide synthase (eNOS). To better understand the mechanisms by which estrogen acutely increases endothelial NO production, proteins of the mitogen activated protein kinase (MAPK) family, ERK, JNK, and p38 were examined to determine their potential role in this process. Bovine aortic endothelial cells (BAEC) were grown to confluence in phenol red-free DMEM F-12, supplemented with 10% fetal calf serum, on 35 cm² dishes. After confluence, cells were starved for 12–24 hours and then exposed to 10 nM 17-estradiol (E₂) for 1–60 minutes. The cells were then lysed and proteins resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. Membranes were then probed with anti-phosphospecific antibodies for ERK, JNK, and p38. Antibodies specific for total ERK and JNK were used to assure even protein loading. The level of ERK phosphorylation rapidly increased in biphasic pattern after E2 exposure: 1 min, 4.3 fold; 5 min, 1 fold; and 10 min, 7.3 fold (max). However, the level of JNK phosphorylation did not increase throughout the time course. The level of p38 phosphorylation increased rapidly to 3.6 fold at 10 minutes. We demonstrate that E2 rapidly activates ERK in a biphasic manner and p38 in a monophasic manner, but has no effect on the activity of JNK. Our data suggest that rapid eNOS activation by estrogen in vascular endothelial cells is predominantly mediated through ERK and p38 pathway. Rapid activation of ERK and p38 resulting in NO production may substantially contribute to the atheroprotective effect of estrogen.     

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca²⁺ leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso–redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca²⁺]_i and oxidative stress), and that therapies restoring nitroso–redox balance in the heart could prevent sudden arrhythmic death.Nitric oxide (NO) is an important regulator of cardiac function via both the activation of cyclic guanosine monophosphate-dependent signaling pathways and direct posttranslational modification of protein thiols (S-nitrosylation) (1). NO derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins (2). In particular, nitrosylation of both skeletal and cardiac muscle ryanodine receptors (RyR1 and RyR2, respectively) alters their release properties, favoring activation (3, 4). Notably, an increase in RyR2 open probability can cause spontaneous SR Ca²⁺ release, which may cause arrhythmias. Recently, it was shown that decreased RyR2 S-nitrosylation (RyR2-SNO) through loss of NOS1, was associated with increased spontaneous SR Ca²⁺ release events in isolated cardiomyocytes, following rapid pacing (5). In a separate study, NOS1 deficiency was shown to decrease spontaneous SR Ca²⁺ sparks and the open probability of RyR2 under resting conditions in cardiomyocytes and lipid bilayers, respectively (6). These studies suggest that NOS1 deficiency has a variable effect on RyR2 function, likely dependent on the underlying physiological state (i.e., rapid heart rate versus quiescence). It remains unknown, however, whether these changes create a substrate for arrhythmogenesis in the intact heart.It is increasingly evident that activities of nitric oxide and reactive oxygen species (ROS) are tightly coupled in cardiomyocytes producing nitroso–redox balance. Elevated ROS production (oxidative stress) is a hallmark of several cardiovascular diseases associated with increased risk of fatal ventricular arrhythmias [e.g., myocardial infarction (MI) and heart failure]. Burger et al. (7) recently demonstrated an increased incidence of ventricular arrhythmias following MI in NOS1-deficient mice. These data suggest that a nitroso–redox imbalance may be arrhythmogenic in the setting of MI. However, the molecular basis of the increased arrhythmogenesis is not known.In the current study, we found that decreased NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release resulting from a combination of decreased RyR2-SNO and increased RyR2 oxidation (RyR2-SOx) [i.e., increased ROS from xanthine oxidoreductase (XOR) activity] and could be suppressed with xanthine oxidoreductase inhibition (i.e., allopurinol) or nitric oxide donors (i.e., GSNO). Notably, we found evidence of NOS1 regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between the nitrosylation and phosphorylation states of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype under oxidative stress.     

Protein kinase C and Gi-protein mediated modulation of cAMP production in different stages of the rat seminiferous epithelium

The modulatory role of protein kinase C (PK-C)- and G_i-protein-mediated signal transduction systems was studied in the cyclic variation of follicle-stimulating hormone (FSH)-stimulated cAMP production of rat seminiferous tubules. FSH (Metrodin, Serono, 30 mg/1) stimulated cAMP production 10-fold (p < 0.01) in a 3 h incubation of 5 mm segments of seminiferous tubules of stages II–VI of the epithelial cycle, but only 2-fold (p < 0.01) in stages VII–VIII. The PK-C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nmol/1) suppressed the FSH effect on cAMP output by 50–70% (p < 0.01) in stages II–VI, but had no effect in stages VII–VIII. If the tubular segments were preincubated for 3 h in the presence of pertussis toxin (PT, 100 μg/1), the FSH-stimulated cAMP production of stages VII-VIII increased by 100–200% (p < 0.01), and now they also became responsive to the TPA suppression. Conversely, no effect of PT was observed in stages II–VI. Cholera toxin (CT, 100 μg/1) and forskolin (Fk, 100 μmol/1) nearly similarly stimulated the cAMP production in both stages studied (about 10-fold, p < 0.01), and TPA and PT potentiated the effects in a non-additive fashion. In conclusion, both G_i-protein and PK-C-mediated mechanisms modulate cAMP production of rat seminiferous tubules. A clear cyclic variation can only be demonstrated in FSH-stimulated cAMP production, but not if the G_s-protein or adenylate cyclase are directly stimulated. Upon FSH stimulation, the low cAMP production in stages VII–VIII is mainly due to the G_i-protein-mediated inhibition. In contrast, the absence (or non-function) of this inhibition mechanism explains the brisk cAMP response to FSH in stages II–VI. PK-C activation suppresses FSH-stimulated cAMP production only if it is not inhibited by the G_i-protein-mediated mechanism (stages II–VI), probably by inhibiting the FSH-receptor-G_s-protein association. It also increases CT and Fk-stimulated cAMP production, in this case inactivating the G_i-protein.     

Nitric oxide is an excitatory modulator in the rostral ventrolateral medulla in rats

Nitric oxide is a messenger molecule having various functions in the brain. Previous studies have reported conflicting results for the roles of nitric oxide in the rostral ventrolateral medulla, a major center that regulates sympathetic and cardiovascular activities. We hypothesized that in this region, nitric oxide may have a biphasic effect on cardiovascular activity. Microinjection of a low dose (1 nmol) of a nitric oxide donor sodium nitroprusside or a cyclic GMP agonist 8-bromocyclic GMP into this area increased arterial pressure, whereas injection of a nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester or a soluble guanylate cyclase inhibitor methylene blue decreased arterial pressure. Microinjection of a high dose (100 nmol) of sodium nitroprusside decreased arterial pressure and inhibited spontaneous respiration with concomitant production of peroxynitrite, a strong cytotoxic oxidant. Increases in arterial pressure caused by microinjection of L-glutamate were inhibited after preinjection of Nomega-nitro-L-arginine methyl ester or methylene blue. Increases in arterial pressure caused by microinjection of sodium nitroprusside (1 nmol) were inhibited after preinjection of a glutamate receptor antagonist kynurenate. These results suggest that low doses of nitric oxide may increase arterial pressure, whereas high doses of nitric oxide may decrease arterial pressure through cytotoxic effects in the rostral ventrolateral medulla. They also indicate that nitric oxide may stimulate neurons both through activation of the nitric oxide cyclic GMP pathway and through modulation of glutamate receptor stimulation, and therefore, increase arterial pressure in rats.