Inhibitory Effects of Oxyradicals on Surfactant Function: Utilizing in Vitro Fenton Reaction

The inhibitory effects of reactive oxygen species (ROS) on the surface tension-lowering abilities of three surfactants were compared: natural lung surfactant (NLS), KL₄ surfactant containing synthetic peptide resembling the hydrophobic/hydrophilic domains of SP-B in an aqueous dispersion of phospholipids, and Survanta? (SUR) containing SP-B and SP-C. The inhibitory concentrations of Fenton reactants (i.e. 0.65 mM FeCl₂, 0.65 mM EDTA, 30 mM H₂O₂), deduced from dose-response plots of FeCl₂ on minimum surface tension (MST) of SUR, were used to assess the Fenton effect on biophysical properties of various surfactants. Neither H₂O₂ (30 mM) nor FeCl₂ with EDTA (both 0.65 mM) alone affected surfactant function, but when mixed together significantly increased (p < 0.01) the MST of SUR compared with KL₄ (p < 0.05) in a FeCl₂ concentration-dependent manner. This effect on NLS was not significant (p= 0.05) at similar phospholipid concentrations. Also, the range of increases in surface adsorption in mN/m at equilibrium surface tension (EST) was 27–40 for SUR, 36–44 for KL₄, and 24–25 for NLS. We speculate that the presence of SP-A and the catalase content in NLS may have protective effects on inactivation of NLS by ROS. We conclude that the in vitro Fenton effect could be a valuable test system for comparing the inactivation range of surfactants by oxyradicals. Accepted for publication: 20 May 1997     

Characterization of Surfactant Subtypes of Beractant and a Synthetic Peptide Containing Surfactant KL4 following Surface Area Cycling and Addition of Fibrinogen

Surfactant is not a homogeneous material and can be separated into subtypes. Subtype conversion is clinically important because it is thought to occur naturally and because surface activity varies depending on the subtype. Fibrinogen, a naturally occurring serum protein, is known to affect this conversion. In this study we studied two surfactants, beractant and KL₄, to examine their subtype characteristics. Surface area cycling, an in vitro method, was used in conjunction with sucrose gradient ultracentrifugation to separate subtypes in both surfactants. Activity, expressed as minimum surface tension of these subtypes, was measured using a pulsating bubble surfactometer. The effect of fibrinogen on subtype conversion and subsequent change in activity was elucidated. Our results indicate that following surface area cycling, beractant and KL₄ have different subtypes and different responses to fibrinogen. Cycling of beractant resulted in two bands, representing a heavy and a light subtype. In the presence of fibrinogen, cycling resulted in two separate heavy subtypes. Cycling of KL₄ surfactant also yielded light and heavy subtypes. However, in the presence of fibrinogen, cycling of KL₄ resulted in ultraheavy subtypes. These ultraheavy subtypes retained minimum surface tension comparable to that of native KL₄ surfactant. We conclude that these two surfactant preparations have different subtype conversions when subjected to surface area cycling and in the presence of fibrinogen. These conversions result in different activities toward lowering surface tension. We speculate that endogenous fibrinogen will also affect these two surfactants differently in vivo and thus affect their clinical effectiveness. Accepted for publication: 14 November 1996     

The influence of pH on surface properties of lung surfactants

Protein-lipid interactions at air-liquid interfaces are dependent on electrostatic charges, cations, anions, distribution of protons, and surface potential, which are influenced by pH changes. All of these factors may affect lung surfactant function. To verify the pH dependence of surface activity of pulmonary surfactant, we studied the in vitro effects of pH and Ca²⁺ on the surface tension-lowering abilities of various surfactants in an oscillating bubble surfactometer. Surface tension measurements were made of mixtures at known pHs or after the replacement of the subphase fluid of surfactant films with buffered saline at various pH values. At the pH range 4.0–7.0, the average equilibrium surface tension (EST)/minimum surface tension (MST) for natural surfactants human amniotic fluid and natural lung surfactant from rabbit lung lavage was 24/2 mN/m. At the same pH range, Exosurf and phospholipids alone had an EST/MST of 44/25 and 44/12 mN/m, respectively. Survanta (SUR) containing SP-B and SP-C and a phospholipid surfactant (KL₄) containing a leucine/lysine peptide had an EST/MST of 29/5 and 36/3, respectively. Alkalinization of the subphase (pH > 7.4) significantly decreased the surface tension-lowering ability of SUR (P < 0.01) and to a lesser extent that of KL₄ surfactant (P < 0.05), but natural lung surfactants were not significantly affected over a pH range of 3–7.5. These data demonstrate that natural surfactants maintain their optimal surface activities over a broader pH range than do the commercial products because of a lack of SP-A. Careful monitoring of the pH for optimal surface activity is recommended when evaluating surfactant function and the effects of specific inhibitors on this function. Offprint requests to: J. D. Antirkhanian, PhD     

Budesonide Down-Regulates Eosinophil Locomotion But Has No Effects on ECP Release or on H2O2 Production

Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H₂O₂) production in the presence of different drug concentrations similar to those obtained at airway level (10⁻⁸ and 10⁻⁷ M) and at blood level (10⁻¹⁰ and 10⁻⁹ M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 μg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H₂O₂ production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10⁻⁸ and 10⁻⁷ M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H₂O₂ production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins. Accepted for publication: 11 February 1999     

Methacholine Dose-Response Slopes from Maximal Bronchial Challenge Tests in Asthmatic Children: Methodological Aspects

To determine whether the slope of a maximal bronchial challenge test (in which FEV₁ falls by over 50%) could be extrapolated from a standard bronchial challenge test (in which FEV₁ falls up to 20%), 14 asthmatic children performed a single maximal bronchial challenge test with methacholine (dose range: 0.097–30.08 μmol) by the dosimeter method. Maximal dose-response curves were included according to the following criteria: (1) at least one more dose beyond a ΔFEV₁≥ 20%; and (2) a MFEV₁≥ 50%. PD₂₀ FEV₁ was calculated, and the slopes of the early part of the dose-response curve (standard dose-response slopes) and of the entire curve (maximal dose-response slopes) were calculated by two methods: the two-point slope (DRR) and the least squares method (LSS) in % ΔFEV₁×μmol⁻¹. Maximal dose-response slopes were compared with the corresponding standard dose-response slopes by a paired Student's t test after logarithmic transformation of the data; the goodness of fit of the LSS was also determined. Maximal dose-response slopes were significantly different (p < 0.0001) from those calculated on the early part of the curve: DRR_20% (91.2 ± 2.7 ΔFEV₁% ·μmol⁻¹) was 2.88 times higher than DRR_50% (31.6 ± 3.4 ΔFEV₁% ·μmol⁻¹), and the LSS_20% (89.1 ± 2.8% ΔFEV₁·μmol⁻¹) was 3.10 times higher than LSS_50% (28.8 ± 1.5% ΔFEV₁·μmol⁻¹). The goodness of fit of LSS_50% was significant in all cases, whereas LSS_20% failed to be significant in one. These results suggest that maximal dose-response slopes cannot be predicted from the data of standard bronchial challenge tests. Accepted for publication: 12 December 1996     

Antioxidant Function of Ambroxol in Mononuclear and Polymorphonuclear Cells in Vitro

This study quantifies the antioxidant function of ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) in vitro. Polymorphonuclear cells (PMN) and mononuclear cells were isolated from the blood of healthy volunteers (n= 46) to determine reactive oxygen species (ROS) by luminol-enhanced chemiluminescence. Ambroxol or the controls N-acetylcysteine (NAC), nacystelyn (NAL), glutathione (GSH), superoxide dismutase (SOD), catalase, and the combination of SOD/catalase were incubated for 1 or 2 h with zymosan-activated cells in vitro using concentrations ranging from 10⁻⁶ to 10⁻³ mol/liter. Reduction of ROS-mediated luminescence was similar within the cell types. Ambroxol (10⁻⁴ mol/liter) reduced ROS about 75% (1-h incubation) and 98% (2-h incubation), respectively (p < 0.001). SOD and SOD/catalase, but not the H₂O₂-catalyzing substances (NAC, NAL, GSH, and catalase), reduced cellular ROS. This indicates that inflammatory cells predominantly generate O⁻ ₂, which can be scavenged by ambroxol. The antioxidant function of ambroxol with increasing incubation time suggests additional cellular antiinflammatory properties of this substance. Our results indicate that good antioxidant function of ambroxol is related mainly to direct scavenger function of reactive oxygen metabolites such as O⁻ ₂. However, an antioxidative effect of ambroxol may also be associated with the reduction of prooxidative metabolism in inflammatory cells. Concluding from this observation, and because of the well known high affinity of ambroxol for lung tissue, ambroxol may be an alternative in antioxidant augmentation therapy, particularly in pulmonary diseases characterized by an overburden of toxic oxygen metabolites. Accepted for publication: 5 December 1996     

Furosemide-Induced Bronchodilation in the Rat Bronchus: Evidence of a Role for Prostaglandins

Pretreatment with inhaled fuorsemide has been shown to protect against bronchoconstrictive stimuli that indirectly activate airway smooth muscle. However, it is controversial as to whether furosemide acts directly on airway smooth muscle. To investigate this we studied the effect of furosemide on both methacholine (MCh)- and serotonin (5-HT)-induced bronchoconstriction in explanted rat airways. Lungs from 21 Sprague-Dawley rats (269 ± 15 g) were excised, inflated with agarose solution at 37°C (1% w/v, 48 ml/kg), embedded in 4% agarose, and refrigerated to gel the agarose. Lung slices (0.5–1.0 mm thick) were cultured overnight at 37°C. Explants were placed on a dissecting video microscope, and airway area was measured with an image analysis system. MCh or 5-HT was administered directly to explanted airways (final concentrations 3.8 × 10⁻⁶ M and 3.8 × 10⁻⁵ M, respectively). Five min later furosemide (3.7 × 10⁻⁵ M or 3.7 × 10⁻⁴ M) was added and airway area monitored 5, 10, 15, 30, and 60 min later. Results were expressed as a percentage of the maximal response. Significant bronchodilation was seen after 30 min in airways preconstricted with MCh and after 15 min in those preconstricted with 5-HT following 3.7 × 10⁻⁴ M furosemide (p < 0.05). 3.7 × 10⁻⁵ M furosemide caused bronchodilation only at 60 min in airways constricted with 5-HT. The effect was blocked by a 30-min incubation of explants with 10⁻⁶ M indomethacin. The furosemide-induced bronchodilation effect was not observed in airways strongly constricted with 3.8 × 10⁻⁵ M MCh. These findings indicate that in the rat at least, furosemide induces a weak bronchodilator effect present only at high doses, which seems to be dependent on the production of prostaglandins. This effect may be relevant to the observed therapeutic action of furosemide in asthmatics. Accepted for publication: 27 September 1996     

Bronchial and Bronchoalveolar Inflammation in Single Early and Dual Responders after Allergen Inhalation Challenge

To characterize the cellular inflammation at the bronchial and bronchoalveolar levels, we evaluated 43 patients with asthma who were sensitized to house dust mites. On 2 consecutive days patients underwent methacholine challenge and allergen bronchial challenge. In addition, 6, 24, or 72 h after allergen challenge, fiberoptic bronchoscopy with bronchial lavage (BL) and bronchoalveolar lavage (BAL) was performed. Patients belonging to the 6-h, 24-h, or 72-h group were divided further into two subgroups: those with isolated early response to allergen (LAR⁻), and those with dual response to allergen (LAR⁺). The percentage of eosinophils and of epithelial cells in BAL fluid was significantly higher in LAR⁺ than in LAR⁻ patients in the 6-h group (p < 0.05, each comparison), but not 24 or 72 h after (p > 0.05, each comparison). Similarly, the proportion of BL eosinophils was also higher in LAR⁺ than in LAR⁻ patients, both in the 6-h and in the 24-h group (p < 0.05, each comparison). In addition, increased proportions of BL neutrophils were present in the LAR⁺ patients belonging to the 24-h group (p < 0.05). Comparing ``proximal' = BL vs ``distal' = BAL data, we found a significantly higher proportion of epithelial cells in BL compared with BAL, in both LAR⁻ and LAR⁺ subjects, either 6, or 24, or 72 h after challenge (p < 0.01, each comparison) and increased percentages of BL neutrophils and eosinophils in LAR⁺ patients (p < 0.05, each comparison), but not in LAR⁻ patients, in the 24-h group. The percentages of BL or BAL macrophages and lymphocytes did not differ significantly among the different patient groups. These data indicate that the development of LAR after allergen inhalation challenge is associated with an early recruitment of eosinophils and with epithelial desquamation in the airways. In addition, after allergen challenge epithelial desquamation is more pronounced in the proximal than in the distal airways, independently of the type of bronchial response. Accepted for publication: 7 January 1997     

Byssinosis: Role of Polymer Length on the Effect of Tannin on the Airway β-Adrenergic Receptor

Tannin, isolated from cotton bracts and implicated in the pathogenesis of byssinosis, inhibits isoproterenol and forskolin-stimulated cAMP release from airway cells in part by decreasing cell surface β-adrenergic receptor number and uncoupling the β-adrenergic receptor from its stimulatory G-protein (G_s) and in part by inhibiting adenylyl cyclase activity. We have hypothesized that cotton tannin, because of its long polymer length, interacts with the hydrophobic binding pocket of the β-adrenergic receptor and alters β-adrenergic receptor binding and G_s coupling. In these studies, tannins of three different polymer lengths and molecular masses were isolated from cotton bracts using sequential Amicon ultrafiltration [molecular mass > 10,000 (YM10 retentate), 1,000–10,000 (YM10 filtrate), and 1,000–5,000 Da (YM2 retentate)]. The YM10 retentate (25 μg/ml) decreased chloride secretion (Jnet = 1.11 ± 0.28 (control) to 0.59 ± 0.18 μEq/cm²·h, p < 0.05, n= 6), decreased cell surface β-adrenergic receptor number (18.0 ± 1.8 (control) to 10.6 ± 0.9 fmol/mg protein, p < 0.02, n= 4), and inhibited forskolin-stimulated cAMP release (5,254 ± 1,290 (control) to 2,968 ± 620 pmol/mg protein, p < 0.01, n= 8). In contrast, neither the YM10 filtrate nor the YM2 retentate had any effect on net chloride secretion, β-adrenergic cell surface receptor number, or forskolin-stimulated cAMP release. We conclude that polymer length is essential for the effect of tannin on the β-adrenergic receptor and on adenylyl cyclase. Accepted for publication: 28 June 1998     

Involvement of Tachykinins in Endotoxin-Induced Airway Hyperresponsiveness

Inhaled endotoxin, lipopolysaccharide (LPS), has been shown to result in bronchial hyperresponsiveness (BHR) to endogenous bronchoconstrictive mediators such as histamine. To determine the role of sensory neuropeptides released from bronchopulmonary C-fibers in LPS-induced BHR, 24 guinea pigs were allocated randomly to the following four groups. Animals in Groups I and IV were challenged with intratracheal instillation of 100 μl of saline vehicle, and those in Groups II and III with 1 mg of LPS (Escherichia coli, 0111:B4) in 100 μl of saline. Groups III and IV also received a high dose capsaicin (HDC) treatment to deplete tachykinins from C-fibers 1–2 weeks prior to the experiment. Animals were anesthetized and paralyzed, and total lung resistance (R_L) and compliance (C_dyn) were measured continuously during the experiment. Dose responses of R_L and C_dyn to histamine (0–8 μg/kg, intravenously) and capsaicin (0–1.6 μg/kg, intravenously), a specific C-fiber stimulant, were obtained prior to and at 1, 2, and 3 h following LPS/saline vehicle challenge. At 2 h after LPS, ΔR_L caused by histamine (8 μg/kg) was significantly higher in Group II (1.145%) than that in Group I (280%; p < 0.05); similarly, ΔR_L caused by capsaicin (1.6 μg/kg) was also increased after LPS (Group I, 107%; Group II, 267%; p < 0.05). Although HDC treatment completely abolished the bronchomotor response to capsaicin in both Groups III and IV, it enhanced the LPS-induced BHR to histamine (8 μg/kg; Group III, 1.834%; p < 0.05). In conclusion, these results suggest that the role of tachykinins in LPS-induced BHR may be dependent upon the type and the route of administration of the bronchoactive substance studied. Accepted for publication: 13 December 1996     

Protecting against peroxynitrite-mediated cytotoxicity in vascular smooth muscle cells via upregulating endogenous glutathione biosynthesis by 3H-1,2-dithiole-3-thione

Peroxynitrite (ONOO⁻) is critically involved in the pathogenesis of cardiovascular diseases. Reaction with glutathione (GSH) was proposed to be a major detoxification pathway of ONOO⁻ in the biological system. This study was undertaken to determine if chemically elevated intracellular GSH affords protection against ONOO⁻-mediated toxicity in vascular cells. Incubation of aortic smooth muscle A10 cells with 3H-1,2-dithiole-3-thione (D3T) led to a concentration- and time-dependent elevation of cellular GSH. Treatment of the cells with D3T also augmented protein and gene expression of γ-glutamylcysteine ligase. To examine the effects of D3T-induced GSH on ONOO⁻-mediated toxicity, we pretreated A10 cells with D3T and then exposed them to either authentic ONOO⁻ or the ONOO⁻ generator, 3-morpholinosydnonimine. We observed that D3T pretreatment of A10 cells resulted in a significant protection against ONOO⁻ cytotoxicity. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of ONOO⁻ cytotoxicity. To further demonstrate the causal involvement of GSH induction in D3T cytoprotection, we cotreated A10 cells with BSO to abolish D3T-induced GSH elevation. BSO cotreatment was found to greatly reverse the protective effects of D3T on ONOO⁻-elicited cytotoxicity. Taken together, our results demonstrate that upregulating GSH biosynthesis by D3T results in a marked protection against ONOO⁻-induced toxicity in vascular cells.     

Atypical Adrenoceptor-mediated Relaxation of Canine Pulmonary Artery Through a Cyclic Adenosine Monophosphate–dependent Pathway

To determine whether functional atypical β-adrenoceptors (β₃-adrenoceptors) are present in pulmonary vascular smooth muscle, we studied isolated canine pulmonary arterial rings under isometric conditions in vitro. Addition of β-adrenoceptor agonists produced a concentration-dependent relaxation of noradrenaline-precontracted tissues, a rank order potency being isoproterenol (1) > salbutamol (0.95) > selective β₃-adrenoceptor agonists, CL 316243 (0.85), and BRL 37344 (0.83). A marked desensitization to salbutamol occurred by pretreatment with salbutamol but not with CL 316243. When β₁-adrenoceptors had been blocked, the relaxant responses to salbutamol were competitively antagonized by the β₂-adrenoceptor antagonist ICI 118551 with a pA₂ value of 7.67 ± 0.21 (mean ± S.E.), but the response to CL 316243 was weekly antagonized by ICI 118551 only at a high concentration of 10⁻⁵ M, where an apparent pA₂ value was 5.24. In contrast, cyanopindolol, a nonselective β-adrenoceptor antagonist, antagonized CL 316243–induced relaxation in a competitive manner with a pA₂ of 6.10 ± 0.11. This pA₂ value was lower than that when salbutamol was used as an agonist (6.69 ± 0.14, p < 0.01). Intracellular 3′,5′-cyclic adenosine monophosphate (cAMP) levels were increased by CL 316243 in a concentration-dependent fashion, an effect that was not altered by ICI 118551. These results suggest that β₃-adrenoceptors may exist in canine pulmonary artery smooth muscle and that stimulation of this atypical receptor causes vasodilation through a cAMP-dependent pathway. Accepted for publication: 17 June 1999     

The arterial site of action of nitric oxide in the neonatal pig lung determined by microfocal angiography

To determine the site of action of inhaled nitric oxide (iNO) in the newborn pig lung, lungs were isolated and perfused at constant flow for microfocal x-ray angiography. Measurements of pulmonary arterial diameters were made on arteries in the 100–2500 μm diameter range under control conditions, during vasoconstriction caused by hypoxia (decreasing PO₂ from ∼120 to ∼50 Torr), or N^ω-nitro-L-arginine methylester (L-NAME 10⁻⁴ M) administration, with or without vasodilation induced by iNO (40 ppm) or by the NO donor S-nitroso-N-acetylpenicillamine (SNAP 5 × 10⁻⁶ M) given intravascularly. Hypoxia caused constriction only in smaller arteries whereas L-NAME constricted arteries throughout the size range studied. iNO dilated the smaller arteries more than the larger arteries under all study conditions. SNAP was used to provide an intravascular source of NO for comparison to iNO. SNAP also dilated smaller arteries more than larger arteries, but it had a significantly greater effect on the large arteries than did iNO. This suggests that differential accessibility of the vascular smooth muscle to NO between sources, air and blood, is a factor in the diameter dependence of the responses. Accepted for publication: 13 March 2001     

Airway Eosinophilic Inflammation and Bronchial Hyperresponsiveness after Allergen Inhalation Challenge in Asthma

Allergen exposure in atopic asthmatic patients is associated with recruitment and activation of eosinophils in the airways. Once activated, eosinophils release toxic products, including the eosinophil cationic protein (ECP), able to damage bronchial structures and to increase bronchial hyperresponsiveness. With this background, the present study was designed to evaluate whether ECP levels in bronchoalveolar lavage (BAL) fluid could reflect, better than BAL eosinophil counts, the cellular activation that follows allergen exposure in atopic asthmatics. Twenty-two atopic patients attended the laboratory on two separate days. On the 1st day, they underwent methacholine (MCh) inhalation challenge to detect the degree of nonspecific bronchial hyperresponsiveness. On the 2nd day, they underwent fiberoptic bronchoscopy and BAL, at baseline or 4–6 h after allergen inhalation challenge. In this latter patient group, MCh challenge was repeated 3–5 h after allergen challenge, 1 h before fiberoptic bronchoscopy. The analysis of the mean baseline FEV₁ values and the degree of bronchial reactivity to MCh (MCh Pd₂₀) on the 1st study day did not demonstrate differences between the two patient groups (p > 0.1, each comparison). In addition, in the allergen-challenged group, MCh Pd₂₀ was decreased significantly after allergen challenge (151.4 μg/ml and 67.6 μg/ml, respectively, before and after challenge; p < 0.05). Evaluation of the different BAL cell types demonstrated that the proportions of eosinophils and epithelial cells were increased significantly in the allergen-challenged group compared with the group evaluated at baseline (p < 0.01 and p < 0.05, respectively). Moreover, ECP levels, corrected by the correspondent albumin levels (ECP/Alb), were higher in the allergen-challenged group compared with the group evaluated at baseline (p < 0.05). In addition, although a positive correlation was demonstrated between BAL eosinophil percentages and ECP/Alb values (r= 0.72, p < 0.05) in the group evaluated at baseline, no links were found between these parameters in the allergen-challenged group (p > 0.1). However, in this latter group, a weak positive correlation was demonstrated between eosinophil percentages and ΔMch, i.e., the increased nonspecific bronchial reactivity, which is observed after allergen challenge (r= 0.55; p < 0.05). Thus, in stable asthmatic patients an ongoing activation of eosinophils parallels their migration, but this eosinophilic inflammation is not strictly related to bronchial reactivity to Mch. By contrast, after allergen inhalation challenge, eosinophil recruitment and activation seem to follow different temporal kinetics, and eosinophilic inflammation may be partially associated with the degree of airway hyperresponsiveness. Accepted for publication: 15 September 1997     

Vildagliptin therapy reduces postprandial intestinal triglyceride-rich lipoprotein particles in patients with type 2 diabetes

Aims/hypothesis We assessed the effects of vildagliptin, a novel dipeptidyl peptidase IV inhibitor, on postprandial lipid and lipoprotein metabolism in patients with type 2 diabetes.Subjects, materials and methods This was a single-centre, randomised, double-blind study in drug-naive patients with type 2 diabetes. Patients received vildagliptin (50 mg twice daily, n=15) or placebo (n=16) for 4 weeks. Triglyceride, cholesterol, lipoprotein, glucose, insulin, glucagon and glucagon-like peptide-1 (GLP-1) responses to a fat-rich mixed meal were determined for 8 h postprandially before and after 4 weeks of treatment.Results Relative to placebo, 4 weeks of treatment with vildagliptin decreased the AUC_0–8h for total trigyceride by 22±11% (p=0.037), the incremental AUC_0–8h (IAUC_0–8h) for total triglyceride by 85±47% (p=0.065), the AUC_0–8h for chylomicron triglyceride by 65±19% (p=0.001) and the IAUC_0–8h for chylomicron triglyceride by 91±28% (p=0.002). This was associated with a decrease in chylomicron apolipoprotein B-48 (AUC_0–8h, −1.0±0.5 mg l⁻¹ h, p=0.037) and chylomicron cholesterol (AUC_0–8h, −0.14±0.07 mmol l⁻¹ h, p=0.046). Consistent with previous studies, 4 weeks of treatment with vildagliptin also increased intact GLP-1, suppressed inappropriate glucagon secretion, decreased fasting and postprandial glucose, and decreased HbA_1c from a baseline of 6.7% (change, −0.4±0.1%, p<0.001), all relative to placebo.Conclusions/interpretation Treatment with vildagliptin for 4 weeks improves postprandial plasma triglyceride and apolipoprotein B-48-containing triglyceride-rich lipoprotein particle metabolism after a fat-rich meal. The mechanisms underlying the effects of this dipeptidyl peptidase IV inhibitor on postprandial lipid metabolism remain to be explored.     

Variations in the four and a half LIM domains 1 gene (<Emphasis Type="Italic">FHL1</Emphasis>) are associated with fasting insulin and insulin sensitivity responses to regular exercise

Aims/hypothesis The expression of the four and a half LIM domains 1 gene (FHL1) is increased in the muscle of individuals who show an improvement in insulin sensitivity index (S _I) after 20 weeks of exercise training. The aim of the present study was to investigate associations between three FHL1 single nucleotide polymorphisms (SNPs) and variables derived from an IVGTT, both in the sedentary state and in response to exercise training, in participants in the HERITAGE Family Study. Materials and methods SNPs were typed using fluorescence polarisation methodology. Analyses were performed separately by sex and in black and white individuals. Results In black participants, no associations were found with any of the SNPs. In white women (n = 207), SNP rs9018 was associated with the disposition index (D _I), which is calculated as S _I generated from the MINMOD program (×10⁻⁴ min⁻¹[μU/ml]⁻¹) multiplied by acute insulin response to glucose (AIR_g; pmol/l × 10 min), and the glucose disappearance index (K _g) training responses (p = 0.016 and p = 0.008, respectively). In white men (n = 222), all SNPs were associated with fasting glucose levels (p ≤ 0.05) and SNP rs2180062 with the insulin sensitivity index (S _I) (p = 0.04) in the sedentary state. Two SNPs were associated with fasting insulin training response. Fasting insulin decreased to a greater extent in carriers of the rs2180062 C allele (p = 0.01) and rs9018 T allele (p = 0.04). With exercise training, S _I (×10⁻⁴ min⁻¹[μU/ml]⁻¹: 0.68 ± 0.20 vs −0.77 ± 0.44, p = 0.046), D _I (319 ± 123 vs –528 ± 260, p = 0.006) and K _g (per 100 min: 0.09 ± 0.04 vs −0.14 ± 0.8, p = 0.03) improved more in the C allele carriers at rs2180062 than in the T allele carriers. Conclusions/interpretation Fasting insulin and S _I responses to exercise training were associated with DNA sequence variation in FHL1 in white men. Whether these associations exist only in white men remains to be investigated. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Guinea Pig Pulmonary Mechanics: Altered Sensitivity to Carbachol by Cadmium and/or Selenium

Male Hartley guinea pigs (480–610 g) were treated intratracheally as follows: saline, cadmium (Cd, 0.3 mg), selenium (Se, 0.3 or 0.06 mg), or Se (0.06 mg) and Cd (0.3 mg) simultaneously. Selenium and Cd were administered as sodium selenite and cadmium chloride, respectively. Twenty-four h later, dynamic lung compliance (C_dyn) and pulmonary resistance (R_p) were measured before (baseline C_dyn and R_p) and after carbachol administration (0.0001, 0.001, 0.01, and 0.1 μmol/kg, intravenously). Results indicated a significant decrease in baseline C_dyn caused by 0.3 mg of Cd, 0.3 mg or 0.06 mg of Se, and 0.3 mg of Cd with 0.06 mg of Se (p < 0.05). A significant increase in baseline R_p due to 0.3 mg of Se was observed (p < 0.05). Carbachol decreased C_dyn significantly below baseline, evident after lower doses of carbachol, in guinea pigs pretreated with 0.3 mg of Se, whereas a significant improvement in C_dyn was seen after 0.0001 μmol/kg carbachol in the group pretreated with Se and Cd simultaneously (p < 0.05) compared with the respective baseline values of the saline-treated group. Similarly, a significant increase in R_p was observed after carbachol in groups pretreated with 0.3 mg of Cd or Se (p < 0.05). Results also indicated a significant increase in large airway constriction caused by Cd and/or Se (p < 0.05). A leftward shift in the carbachol dose-response curve indicated increased sensitivity to carbachol in Cd- and/or Se-pretreated guinea pigs. Accepted for publication: 14 March 1997     

Arrhythmogenic peroxynitrite-induced alterations in mammalian heart contractility and its prevention with quercetin-filled liposomes

The aim of the present study was to evaluate the effects of quercetin-filled phosphatidylcholine liposomes (PCLs) on peroxynitrite (ONOO⁻)-induced cardiac arrhythmias. Experiments were done using different experimental models, including isolated rat papillary muscle, Langendorff perfused rat hearts, and anesthetized animals. Being exogenously applied in a concentration greater than 50 μM, ONOO⁻, caused inhibition of isometric twitch amplitude in isolated papillary muscles and led to an appearance of arrhythmias. Decomposed ONOO⁻ had no similar effects and reversibly increased twitch amplitude. Authentic nitric oxide (NO, 100 μM) did not produce arrhythmias and had no significant effect on twitch amplitude. Verapamil and ruthenium red were with-out effect on ONOO⁻-induced arrhythmias, whereas tetrodotoxin and nicorandil effectively prevented arrhythmias development. Ouabain increased the arrhythmogenic effect of ONOO⁻. ONOO⁻ significantly decreased coronary perfusion pressure (CPP) and mean left-ventricular pressure (MLVP) in the Langendorff perfused rat heart and produced severe arrhythmias. Authentic nitric oxide (NO) decreased CPP and MLVP insignificantly and resulted in a low incidence of arrhythmias. The NO donor SIN-1 in doses greater than 50 μM led to the appearance of low-incidence arrhythmias in anesthetized rats. Intraventricular injection of ONOO⁻ promotes the appearance of a high incidence of arrhythmias in anesthetized rats and decreased MLVP. PCLs filled with the antioxidant quercetin restored normal cardiac contractility in both isolated tissues and anesthetizes animals. In conclusion, we hypothesized that ONOO⁻, but not its decomposed products, can initiate membrane lipid peroxidation and damage the phospholipid environment of ionic channels in myocardial cell plasma membranes inducing abnormal cardiac action potentials, arrhythmogenesis, and contractile dysfunction. Quercetin-filled PCL provide reliable protection against peroxynitrite-induced myocardial injury in isolated cardiac tissues and anesthetized animals primarily as a result of the decomposition of endogenously formed ONOO⁻.     

Cell Volume Regulation During Hyperosmotic Shrinkage Is Mediated by Na+/K+-ATPase and Na+–K+–2Cl− Cotransporter in Necturus Gastrics Surface Epithelial Cells

Cell volume regulation was investigated in gastric surface epithelial cells during hypertonic conditions. Isolated Necturus antral mucosa was perfused on the serosal side with Ringer's solution (pH 7.25, 95%O₂/5%CO₂) and on the mucosal side successively with 150–500 mM NaCl. Amiloride, ouabain, and bumetanide were used to experimentally inhibit Na⁺/H⁺, Na⁺/K⁺ ATPase or Na⁺–K⁺–2Cl^– ion transporters. Intracellular sodium activity and cell volume changes were measured with liquid sensor microelectrodes. The increase in intracellular sodium activity caused by luminal hyperosmolar exposure was mainly due to cell shrinkage. Inhibition of Na⁺/K⁺ ATPase or Na⁺–K⁺–2Cl^– cotransporter increased hyperosmotic cell shrinkage (−52 ± 5%, −85 ± 19%, and −77 ± 9% for control, ouabain, and bumetanide, respectively). Inhibition of Na⁺/K⁺ ATPase increased intracellular sodium activity (from 18 ± 4 to 52 ± 12 mM). Cell volume regulation in gastric epithelial surface cells during mucosal hyperosmolar exposure is maintained by the basolateral Na⁺–K⁺–2Cl^– cotransporter, while Na⁺/K⁺ ATPase maintains sodium balance, but Na⁺/H⁺ antiport seems to have a less important role. This work was supported by the Research Foundation of Helsinki University Central Hospital (EVO), Academy of Finland, Antti and Jenny Wihuri Foundation, and Biomedicum Research Foundation, Helsinki, Finland. The authors thank Mrs Paula Kokko for excellent technical assistance.