Reactive oxygen species, PKC-beta1, and PKC-zeta mediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells

Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEGF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22(phox), a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-beta(1) with the specific pharmacological inhibitor LY-333531 or inhibition of PKC-zeta with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-beta(1), or PKC-zeta inhibition. In normal glucose (5.6 mM), overexpression of p22(phox) or constitutively active PKC-zeta enhanced VEGF expression. Hypoxia inducible factor-1alpha protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-beta(1) and -zeta, play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells.     

Role of PKC and TGF-beta receptor in glucose-induced proliferation of smooth muscle cells

The role of protein kinase C (PKC) and transforming growth factor (TGF)-beta in the proliferation of vascular smooth muscle cells (SMCs) under a high glucose condition was investigated. [3H]-thymidine incorporation under 20 mM glucose was significantly accelerated compared with that under 5.5 mM glucose, and this increase was inhibited by an anti-TGF-beta antibody or a PKC-beta specific inhibitor, LY333531. The amount of active and total TGF-beta1 in the conditioned media did not differ between 5.5 and 20 mM glucose. However, the expression of TGF-beta receptor type II under 20 mM glucose was significantly increased, but that of the TGF-beta receptor type I was not. This increased expression of the TGF-beta receptor type II was prevented by LY333531. These observations suggest that the increased expression of the TGF-beta receptor type II via PKC-beta plays an important role in the accelerated proliferation of SMCs under a high glucose condition, leading to the development of diabetic macroangiopathy.     

Vascular-dependent effects of elevated glucose on postganglionic sympathetic neurons

Perivascular sympathetic nerves are important determinants of vascular function that are likely to contribute to vascular complications associated with hyperglycemia and diabetes. The present study tested the hypothesis that glucose modulates perivascular sympathetic nerves by studying the effects of 7 days of hyperglycemia on norepinephrine (NE) synthesis [tyrosine hydroxylase (TH)], release, and uptake. Direct and vascular-dependent effects were studied in vitro in neuronal and neurovascular cultures. Effects were also studied in vivo in rats made hyperglycemic (blood glucose >296 mg/dl) with streptozotocin (50 mg/kg). In neuronal cultures, TH and NE uptake measured in neurons grown in high glucose (HG; 25 mM) were less than that in neurons grown in low glucose (LG; 5 mM) (P < 0.05; n = 4 and 6, respectively). In neurovascular cultures, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release from neurovascular cultures grown in HG (1.8 ± 0.2%; n = 5) was greater than that from cultures grown in LG (0.37 ± 0.28%; n = 5; P < 0.05; unpaired t-test). In vivo, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release in hyperglycemic animals (9.4 + 1.1%; n = 6) was greater than that in control animals (5.39 + 1.1%; n = 6; P < 0.05; unpaired t-test). These data identify a novel vascular-dependent effect of elevated glucose on postganglionic sympathetic neurons that is likely to affect the function of perivascular sympathetic nerves and thereby affect vascular function.     

PKC-delta-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase

Oxidative stress is thought to be one of the causative factors contributing to insulin resistance and type 2 diabetes. Previously, we showed that reactive oxygen species (ROS) production is significantly increased in adipocytes from high-fat diet-induced obese and insulin-resistant mice (HF). ROS production was also associated with the increased activity of PKC-delta. In the present studies, we hypothesized that PKC-delta contributes to ROS generation and determined their intracellular source. NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) reduced ROS levels by 50% in HF adipocytes, and inhibitors of NO synthase (L-NAME, 1 mM), xanthine oxidase (allopurinol, 100 microM), AGE formation (aminoguanidine, 10 microM), or the mitochondrial uncoupler (FCCP, 10 microM) had no effect. Rottlerin, a selective PKC-delta inhibitor, suppressed ROS levels by approximately 50%. However, neither GO-6976 nor LY-333531, effective inhibitors toward conventional PKC or PKC-beta, respectively, significantly altered ROS levels in HF adipocytes. Subsequently, adenoviral-mediated expression of wild-type PKC-delta or its dominant negative mutant (DN-PKC-delta) in HF adipocytes resulted in either a twofold increase in ROS levels or their suppression by 20%, respectively. In addition, both ROS levels and PKC-delta activity were sharply reduced by glucose depletion. Taken together, these results suggest that PKC-delta is responsible for elevated intracellular ROS production in HF adipocytes, and this is mediated by high glucose and NADPH oxidase.     

Age-dependent activation of PKC isoforms by angiotensin II in the proximal nephron

ANG II increases fluid absorption in proximal tubules from young rats more than those from adult rats. ANG II increases fluid absorption in the proximal nephron, in part, via activation of protein kinase C (PKC). However, it is unclear how age-related changes in ANG II-induced stimulation of the PKC cascade differ as an animal matures. We hypothesized that the response of the proximal nephron to ANG II decreases as rats mature due to a reduction in the amount and activation of PKC rather than a decrease in the number or affinity of ANG II receptors. Because PKC translocates from the cytosol to the membrane when activated, we first measured PKC activity in the soluble and particulate fractions of proximal tubule homogenates exposed to vehicle or 10(-10) M ANG II from young (26 +/- 1 days old) and adult rats (54 +/- 1 days old). ANG II increased PKC activity to the same extent in homogenates from young rats (from 0.119 +/- 0.017 to 0.146 +/- 0.015 U/mg protein) (P < 0.01) and adult rats (from 0.123 +/- 0.020 to 0.156 +/- 0.023 U/mg protein) (P < 0.01). Total PKC activity did not differ between groups (0.166 +/- 0.018 vs. 0.181 +/- 0.023). We next investigated whether activation of the alpha-, beta-, and gamma-PKC isoforms differed by Western blot. In homogenates from young rats, ANG II significantly increased activated PKC-alpha from 40.2 +/- 6.5 to 60.2 +/- 9.5 arbitrary units (AU) (P < 0.01) but had no effect in adult rats (46.1 +/- 5.1 vs. 48.5 +/- 8.2 AU). Similarly, ANG II increased activated PKC-gamma in proximal tubules from young rats from 47.9 +/- 13.2 to 65.6 +/- 16.7 AU (P < 0.01) but caused no change in adult rats. Activated PKC-beta, however, increased significantly in homogenates from both age groups. Specifically, activated PKC-beta increased from 8.6 +/- 1.4 to 12.2 +/- 2.1 AU (P < 0.01) in homogenates from nine young rats and from 19.0 +/- 5.5 to 25.1 +/- 7.1 AU (P < 0.01) in homogenates from 12 adult rats. ANG II did not alter the amount of soluble PKC-alpha, -beta, and -gamma significantly. The total amount of PKC-alpha and -gamma did not differ between homogenates from young and adult rats, whereas the total amount of PKC-beta was 59.7 +/- 10.7 and 144.9 +/- 41.8 AU taken from young and adult rats, respectively (P < 0.05). Maximum specific binding and affinity of ANG II receptors were not significantly different between young and adult rats. We concluded that the primary PKC isoform activated by ANG II changes during maturation.     

Antiproliferative effect of elevated glucose in human microvascular endothelial cells

Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1–14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10–50 μM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular pathophysiology associated with diabetic microangiopathy. J. Cell. Biochem. 71:491–501, 1998. © 1998 Wiley-Liss, Inc.     

Influence of alpha-tocopherol on prostacyclin synthesis by rat's arterial and myometrial tissues

Influence of alpha-tocopherol on PGI2 synthesis by rat arterial and myometrial tissues was investigated using a rat platelet antiaggregatory bioassay. Chronic administration of alpha-tocopherol to female rats (10 mg kg-1 day-1 s.c. for 14 days) significantly increased ex-vivo PGI2 synthesis by the arterial tissue from 12.7 +/- 0.3 (control, mean +/- s.e.m) to 17.2 +/- 0.4 ng PGI2 mg-1 wet tissue and by the myometrial tissue (in proestrus) from 1.1 +/- 0.07 (control) to 1.85 +/- 0.1 ng PGI2 mg-1 wet tissue (P less than 0.05, n = 6). alpha-tocopherol (5 mg kg-1 day-1 for 14 days) did not stimulate PGI2 to any significant level. Pretreatment of male rat arterial tissue with alpha-tocopherol (0.02, 0.1 or 0.2 mM) in vitro increased PGI2 synthesis in a dose-dependent manner. At a dose of 0.2 mM it increased PGI2 synthesis from 13.70 +/- 0.70 (control) to 22.6 +/- 1.4 ng PGI2 mg-1 wet tissue (P less than 0.1, n = 6). Pre-treatment of 14-day pregnant rat myometrium with alpha-tocopherol 0.2 and 0.4 mM significantly increased PGI2 synthesis from 1.2 +/- 0.06 (control) to 1.90 +/- 0.12 and 2.1 +/- 0.1 ng PGI2 mg-1 wet tissue, respectively (P less than 0.05, n = 6). The results indicate that the ability of alpha-tocopherol to stimulate PGI2 synthesis may partly contribute towards better understanding of the mechanisms that underly the protective effect of alpha-tocopherol against experimentally induced decreases in coronary flow and intravascular coagulations in some mammals. Furthermore adequate intake of alpha-tocopherol during pregnancy may enhance uterine blood flow and ensure adequate foetal nutrition.     

PKC-beta1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants

Using monolayers of human intestinal (Caco-2) cells, we found that oxidants and ethanol damage the cytoskeleton and disrupt barrier integrity; epidermal growth factor (EGF) prevents damage by enhancement of protein kinase C (PKC) activity and translocation of the PKC-beta1 isoform. To see if PKC-beta1 mediates EGF protection, cells were transfected to stably over- or underexpress PKC-beta1. Transfected monolayers were preincubated with low or high doses of EGF (1 or 10 ng/ml) or 1-oleoyl-2-acetyl-sn-glycerol [OAG; a PKC activator (0.01 or 50 microM)] before treatment with oxidant (0.5 mM H(2)O(2)). Only in monolayers overexpressing PKC-beta1 (3.1-fold) did low doses of EGF or OAG initiate protection, increase tubulin polymerization (assessed by quantitative immunoblotting) and microtubule architectural integrity (laser scanning confocal microscopy), maintain normal barrier permeability (fluorescein sulfonic acid clearance), and cause redistribution of PKC-beta1 from cytosolic pools into membrane and/or cytoskeletal fractions (assessed by immunoblotting), thus indicating PKC-beta1 activation. Antisense inhibition of PKC-beta1 expression (-90%) prevented these changes and abolished EGF protection. We conclude that EGF protection against oxidants requires PKC-beta1 isoform activation. This mechanism may be useful for development of novel therapies for the treatment of inflammatory gastrointestinal disorders including inflammatory bowel disease.     

Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NO(x)

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110-130 days gestation; term = 145 +/- 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n = 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO(2)-NO(3) (NO(x)). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT < or = FOL < PREG (P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG (P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls (P < 0.01). Circulating NO(x) levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 +/- 1.1 microM in LUT to 12 +/- 4, 22 +/- 3, and 41 +/- 3 microM at 110, 120, and 130 days gestation (P < 0.01). Systemic NO(x) levels in singleton (12.5 +/- 1.6 microM) were less (P < 0.01) than in multiple (twin 27.6 +/- 6.5 microM; triplet = 46 +/- 10 microM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NO(x) levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.     

D-Glyceraldehyde causes production of intracellular peroxide in pancreatic islets, oxidative stress, and defective beta cell function via non-mitochondrial pathways

D-Glyceraldehyde (D-GLYC) is usually considered to be a stimulator of insulin secretion but theoretically can also form reactive oxygen species (ROS), which can inhibit beta cell function. We examined the time- and concentration-dependent effects of D-GLYC on insulin secretion, insulin content, and formation of ROS. We observed that a 2-h exposure to 0.05-2 mM D-GLYC potentiated glucose-stimulated insulin secretion (GSIS) in isolated Wistar rat islets but that higher concentrations inhibited GSIS. A 24-h exposure to 2 mm D-GLYC inhibited GSIS, decreased insulin content, and increased intracellular peroxide levels (2.14 +/- 0.31-fold increase, n = 4, p < 0.05). N-Acetylcysteine (10 mM) prevented the increase in intracellular peroxides and the adverse effects of d-GLYC on GSIS. In the presence of 11.1 but not 3.0 mm glucose, koningic acid (10 microM), a specific glyceraldehyde-3-phosphate dehydrogenase inhibitor, increased intracellular peroxide levels (1.88 +/- 0.30-fold increase, n = 9, p < 0.01) and inhibited GSIS (control GSIS = p < 0.001; koningic acid GSIS, not significant). To determine whether oxidative phosphorylation was the source of ROS formation, we cultured rat islets with mitochondrial inhibitors. Neither rotenone or myxothiazol prevented D-GLYC-induced increases in islet ROS. Adenoviral overexpression of manganese superoxide dismutase also failed to prevent the effect of D-GLYC to increase ROS levels. These observations indicate that exposure to excess D-GLYC increases reactive oxygen species in the islet via non-mitochondrial pathways and suggest the hypothesis that the oxidative stress associated with elevated D-GLYC levels could be a mechanism for glucose toxicity in beta cells exposed chronically to high glucose concentrations.     

Mechanisms of angiotensin II-enhanced connecting tubule glomerular feedback

Increasing Na delivery to the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation, a process we call CNT glomerular feedback (CTGF). Angiotensin II (ANG II) in the CNT lumen enhances CTGF via PKC. We hypothesized that luminal ANG II stimulates CTGF via activation of protein kinase C (PKC), NADPH oxidase 2 (NOX2), and enhanced production of superoxide (O(2)(-)). Rabbit Af-Arts and adherent CNTs were microdissected and microperfused in vitro. Dilation of the Af-Art was induced by increasing luminal CNT NaCl from 0 to 5, 10, 30, 45, and 80 mM, and the concentration of NaCl that elicited a half-maximal response (EC(50)) was calculated. Compared with vehicle, adding ANG II (10(-9) M) to the CNT lumen reduced EC(50) from 37 ± 3 to 14 ± 1 mM (P < 0.001), indicating ANG II potentiates CTGF. In the presence of ANG II, the O(2)(-) scavenger tempol (10(-4) M) increased EC(50) from 20 ± 4 to 41 ± 3 mM (P < 0.01), the NOX inhibitor apocynin (10(-5) M) increased EC(50) from 17 ± 2 to 39 ± 4 mM (P < 0.01), and the specific NOX2 inhibitor gp91ds-tat (10(-5) M) increased EC(50) from 19 ± 2 to 34 ± 2 mM (P < 0.01). However, tempol, apocynin, and gp91ds-tat had no effect on CTGF in the absence of ANG II. Compared with vehicle, the PKC activator PMA (2 × 10(-7) M) decreased EC(50) from 35 ± 2 to 14 ± 1 (P < 0.001). In the presence of PMA, tempol increased EC(50) from 14 ± 2 to 35 ± 2 mM (P < 0.01). We conclude the PKC/NOX2/O(2)(-) pathway mediates the enhancement of CTGF by luminal ANG II but it does not participate in CTGF in the absence of ANG II.     

Effects of antioxidants on the development of bovine IVM/IVF embryos in various concentrations of glucose

This study was undertaken to determine the effects of glucose, antioxidants and different oxygen tensions on the development of bovine embryos cultured in modified synthetic oviduct fluid (m-SOF) medium. In vitro matured (IVM) and fertilized (IVF) oocytes were incubated for 48 h. Embryos reaching at least the 4-cell stage were selected for further culture under various conditions for 6 d. Supplementing the m-SOF media with 4.5 mM glucose resulted in a significantly lower (P < 0.01) embryo developmental rate (21%; Day 8) than was obtained with 1.5 mM glucose (58%; Day 8) or no glucose (53%; Day 8). Antioxidants such as SOD, catalase and mannitol had no positive effect on embryo development in m-SOF medium supplemented with 1.5 mM glucose. However, in m-SOF medium supplemented with 4.5 mM glucose, SOD and mannitol significantly (P < 0.05) improved embryo development: SOD increased the developmental rate from 19 to 35% (Day 8), while mannitol increased it from 13 to 30% (Day 8). Low oxygen concentration improved embryo development significantly (P < 0.05) in m-SOF medium supplemented with 4.5 mM glucose (low O2: 31% vs high O2: 14%; Day 8) but not 0 mM glucose (low O2: 58% vs high O2: 55%; Day 8). Our data suggest that low concentration of glucose during culture of bovine embryos is beneficial, and that generation of free oxygen radicals is partly caused by a high concentration of glucose in the medium.     

Role of protein kinase C in alpha(1)-adrenergic regulation of a(Na)(i) in guinea pig ventricular myocytes

We investigated the role of protein kinase C (PKC) in alpha(1)-adrenergic regulation of intracellular Na(+) activity (a(Na)(i)) in single guinea pig ventricular myocytes. a(Na)(i) and membrane potentials were measured with the Na(+)-sensitive indicator sodium-binding benzofuran isophthalate and conventional microelectrodes, respectively, at room temperature (24-26 degrees C) while myocytes were stimulated at a rate of 0.25-0.3 Hz. The PKC activator 4beta-phorbol 12-myristate 13-acetate (PMA) decreased a(Na)(i) in a concentration-dependent manner. PMA (100 nM) produced a maximal decrease in a(Na)(i) of 1.5 mM from 6.5 +/- 0.4 to 5.0 +/- 0.4 mM (means +/- SE, n = 12, P < 0.01). The PMA concentration required for a half-maximal decrease in a(Na)(i) was 0.46 +/- 0.13 nM (n = 3, P < 0.01). An inactive phorbol, 4alpha-phorbol 12-myristate 13-acetate, did not decrease a(Na)(i). The decrease caused by PMA could be blocked by the PKC inhibitors staurosporine and bisindolylmaleimide I (GF-109203X). Stimulation of the alpha(1)-adrenoceptor with 50 microM phenylephrine decreased a(Na)(i) from 6.1 +/- 0.3 to 4.6 +/- 0.3 mM (n = 11, P < 0.01). The decrease in a(Na)(i) produced by phenylephrine was blocked by pretreatment with staurosporine, GF-109203X, or PMA. The decrease in a(Na)(i) produced by PMA was not prevented by pretreatment with tetrodotoxin but was blocked by pretreatment with strophanthidin or high extracellular K(+) concentration. The results suggest that alpha(1)-adrenergic receptor activation results in a decrease in a(Na)(i) via PKC-induced stimulation of the Na(+)-K(+) pump in cardiac myocytes.     

Mixed tocopherol preparation is superior to alpha-tocopherol alone against hypoxia-reoxygenation injury

Hypoxia-reoxygenation (H-R) is associated with alterations in oxidant-antioxidant balance and L-arginine-nitric oxide system. Tocopherols decrease the activity of reactive oxygen species (ROS) and yet are not beneficial in clinical trials. It has been proposed that mixed tocopherols as found in nature may be more tissue protective than alpha-tocopherol alone found in commercial preparations. We compared the effect of a mixed tocopherol preparation with that of alpha-tocopherol alone on superoxide dismutase (SOD) activity and iNOS expression in cultured myocytes exposed to H-R. Myocytes from Sprague-Dawley rat hearts were subjected to hypoxia for 24 h followed by reoxygenation for 3 h H-R. Parallel groups of myocytes were pretreated with alpha-tocopherol alone or a mixed-tocopherol preparation (containing alpha-, gamma-, and delta-tocopherols) (50 microM) for 30 min. H-R resulted in myocyte injury (determined by LDH release), a decrease in SOD activity and an upregulation of iNOS expression/activity. Both tocopherol preparations attenuated cell injury and markedly decreased the effects of H-R on SOD activity and iNOS expression/activity (all P < 0.05 vs H-R group, n = 5). However, mixed-tocopherol preparation was much superior to alpha-tocopherol in terms of myocyte protection from the adverse effect of H-R (P < 0.05). Lack of efficacy of commercial tocopherol preparations in clinical trials may reflect absence of gamma- and delta-tocopherols.     

Intestinal water absorption from select carbohydrate solutions in humans.

Eight men positioned a triple-lumen tube in the duodenojejunum. By use of segmental perfusion, 2, 4, 6, or 8% solutions of glucose (111-444 mM), sucrose (55-233 mM), a maltodextrin [17-67 mM, avg. chain length = 7 glucose units (7G)], or a corn syrup solid [40-160 mM, avg. chain length = 3 glucose units (3G)] were perfused at 15 ml/min for 70 min after a 30-min equilibration period. All solutions were made isotonic with NaCl, except 6 and 8% glucose solutions, which were hypertonic. An isotonic NaCl solution was perfused as control. Water absorption (range: 9-15 ml.h-1.cm-1) did not differ for the 2, 4, and 6% CHO solutions but was greater (P < 0.05) than absorption from control (3.0 +/- 2.2 ml.h-1.cm-1). The 8% glucose and 3G solutions reduced (P < 0.05) net water flux compared with their 2, 4, and 6% solutions, but 8% sucrose and 8% 7G solutions promoted water absorption equivalent to lower CHO concentrations. Water absorption was independent of [Na+] in the original solution. In the test segment, 1) Na+ flux correlated with net water flux (r = 0.72, P < 0.01), K+ (r = 0.78, P < 0.01), and [Na+] (r = 0.68, P < 0.001); 2) Na+ absorption occurred at luminal [Na+] as low as 50 mM; 3) glucose transport increased linearly over the luminal concentration range of 40-180 mM; and 4) net water flux was similar over a range of glucose-to-Na+ concentration ratios of 0.4:1 to 3.5:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Supplementation of alpha-tocopherol improves cardiovascular risk factors via the insulin signalling pathway and reduction of mitochondrial reactive oxygen species in type II diabetic rats

This study determined the effects of alpha- and gamma-tocopherol supplementation on metabolic control and oxidative stress in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Blood glucose, haemoglobin A1c (HbA1c), urinary protein, plasma free fatty acid, triacylglycerol and plasminogen activator inhibitor-1 (PAI-1) levels in OLETF rats were significantly higher than in non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats. Alpha-tocopherol inhibited the increase in urinary protein, blood glucose, HbA1c and PAI-1 levels, but gamma-tocopherol did not. Plasma and hepatic lipid peroxidation and hepatic steatosis were increased in OLETF rats. alpha-Tocopherol decreased lipid peroxidation. Mitochondrial reactive oxygen species production and uncoupling protein 2 (UCP2) expression were significantly increased in the heart and aorta of OLETF rats compared with LETO rats. Endothelial NO synthase and aortic nitrotyrosine were increased in OLETF rats. In contrast, the expression of phosphorylated vasodilator-stimulated phosphoprotein and glucose transporter 4 in the aorta was significantly decreased in OLETF rats. These abnormalities were reversed by alpha-tocopherol. These findings suggest that alpha-tocopherol may prevent cardiovascular tissues from oxidative stress and insulin signalling disorder resulting from diabetes mellitus.     

Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells

We determined the effect of oxygen [approximately 100 Torr (normoxia) and approximately 30-40 Torr (hypoxia)] on functions of endothelial nitric oxide (NO) synthase (NOS-3) and its negative regulator caveolin-1 in ovine fetal and neonatal lung microvascular endothelial cells (MVECs). Fetal NOS-3 activity, measured as NO production with 0.5-0.9 microM 4-amino-5-methylamino-2,7-difluorofluorescein, was decreased in hypoxia by 14.4% (P < 0.01), inhibitable by the NOS inhibitor N-nitro-L-arginine, and dependent on extracellular arginine. Caveolar function, assessed as FITC-BSA (160 microg/ml) endocytosis, was decreased in hypoxia by 13.5% in fetal and 22.8% in neonatal MVECs (P < 0.01). NOS-3 and caveolin-1 were physically associated, as demonstrated by coimmunoprecipitation and colocalization, and functionally associated, as shown by cross-activation of endocytosis, by their specific antibodies and activation of NOS by albumin. Caveolin peptide, containing the sequence for the PKC phosphorylation site of caveolin, and caveolin antiserum against the site increased NO production and endocytosis by 12.3% (P < 0.05) and 16% (P < 0.05), respectively, in normoxia and increased endocytosis by 25% (P < 0.001) in hypoxia. PMA decreased NO production in normoxia and hypoxia by 19.32% (P < 0.001) and 11.8% (P < 0.001) and decreased endocytosis in normoxia by 20.35% (P < 0.001). PKC kinase activity was oxygen sensitive, and threonine phosphorylation was enhanced in hypoxia. Pertussis toxin increased caveolar and NOS functions. These data support our hypothesis that increased Po(2) at birth promotes dissociation of caveolin-1 and NOS-3, with an increase in their activities, and that PKC and an oxygen-sensitive cell surface G protein-coupled receptor regulate caveolin-1 and NOS-3 interactions in fetal and neonatal lung MVECs.     

Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are decreased in an experimental model of severe respiratory distress syndrome in preterm lambs

Respiratory distress syndrome (RDS) secondary to preterm birth and surfactant deficiency is characterized by severe hypoxemia, lung injury, and impaired production of nitric oxide (NO) and vascular endothelial growth factor (VEGF). Since hypoxia-inducible factors (HIFs) mediate the effects of both NO and VEGF in part through regulation by prolyl-hydroxylase-containing domains (PHDs) in the presence of oxygen, we hypothesized that HIF-1alpha and -2alpha in the lung are decreased following severe RDS in preterm neonatal lambs. To test this hypothesis, fetal lambs were delivered at preterm gestation (115-day gestation, term = 145 days; n = 4) and mechanically ventilated for 4 h. Lambs developed respiratory failure characterized by severe hypoxemia despite treatment with mechanical ventilation with high inspired oxygen concentrations. Lung samples were compared with nonventilated control animals at preterm (115-day gestation; n = 3) and term gestation (142-day gestation; n = 3). We found that HIF-1alpha protein expression decreased (P < 0.05) and PHD-2 expression increased (P < 0.005) at birth in normal term animals before air breathing. Compared with age-matched controls, HIF-1alpha protein and HIF-2alpha protein expression decreased by 80% and 55%, respectively (P < 0.005 for each) in preterm lambs with RDS. Furthermore, VEGF mRNA was decreased by 40%, and PHD-2 protein expression doubled in RDS lambs. We conclude that pulmonary expression of HIF-1alpha, HIF-2alpha, and the downstream target of their regulation, VEGF mRNA, is impaired following RDS in neonatal lambs. We speculate that early disruption of HIF and VEGF expression after preterm birth and RDS may contribute to long-term abnormalities in lung growth, leading to bronchopulmonary dysplasia.     

Ischemia and reperfusion of liver induces eNOS and iNOS expression: effects of a NO donor and NOS inhibitor

The aim of this study was to investigate the role of nitric oxide (NO) in hepatic ischemia-reperfusion (I/R) injury in rats. Immunohistochemistry was used to examine the protein expression of endothelial and inducible nitric oxide synthases (eNOS, iNOS) and nitrotyrosine after I/R challenges to the liver, and blood levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), hydroxyl radical and NO were measured before ischemia and after reperfusion. Ischemia was induced by occlusion of the common hepatic artery and portal vein for 40 min, followed by reperfusion for 90 min. Reperfusion of the liver induced a significant increase in the blood concentrations of AST, ALT, LDH (n = 8; P < 0.001), hydroxyl radical (n = 8; P < 0.001) and NO (n = 8; P < 0.01). The eNOS, iNOS, nitrotyrosine, SOD1 and SOD2 protein expression was also found to increase significantly after reperfusion (n = 3). Administration of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (n = 8) had a protective effect on the I/R-related injury, but the NO donor L-arginine (L-Arg) (n = 8) potentiated the damage caused by I/R. These results suggest that reperfusion of the liver induces expression of NOS, which is related to the elevation of blood NO. The increase in hydroxyl radical concentration was accompanied by an increase in antioxidant enzyme expression (SOD1 and SOD2), and an increase in nitrotyrosine expression was also observed, reflecting the increased production of NO and oxygen radicals. We concluded from the protective effect of L-NAME and the potentiation by L-Arg that NOS expression and increases in NO and hydroxyl radical production have deleterious effects on the response to I/R in the liver.