Acute autonomic effects of vitamins and fats in male smokers.

BACKGROUND/OBJECTIVES: Vitamins can help improve cardiovascular control. In contrast, smoking works in the opposite fashion, reducing the baroreflex control of heart rate (HR) possibly via oxidative stress. High-fat challenges also impair cardiovascular regulation. Whether vitamins have acute beneficial effects on the baroreflex control of HR in smokers is unclear. SUBJECTS/METHODS: A randomized, placebo-controlled crossover study in 30 male smokers (34.2+/-6.9 years). Interventions were: (1) moderate (vitamin C (300 mg) and E (75 IU) and folic acid (1 mg)); (2) high doses of vitamins (vitamin C (2 g) and E (800 IU), and folic acid (5 mg)); or, (3) placebo. Vitamins were ingested with cream (a high-fat challenge) or milk (low-fat control). Four hours later, blood was withdrawn and radial pulse wave forms recorded via tonometry. Spontaneous beat-to-beat variations in HR and systolic blood pressure (SBP) were analysed by spectral analysis techniques and sympathovagal control of HR and baroreflex sensitivity (BRS) were assessed. RESULTS: High doses of vitamins increased plasma vitamin C, E and folic acid levels (P<0.05) with no change in SBP, HR or BRS (P>0.05, analysis of variance). Plasma vitamin levels did not correlate with any cardiovascular parameters. Moderate vitamins increased the vagal control of HR (+23%; P<0.05) and cream led to small increases (P<0.05) in SBP (+2 mm Hg) and HR (+2 beats min(-1)) with no change in BRS. CONCLUSIONS: In male smokers, circulating antioxidants had no effect on BRS and minor effects on the cardiovascular system were seen following acute fat and vitamin ingestion.

Arterial stiffness, endothelial function and microcirculatory reactivity in healthy young males.

Large (C1) and small (C2) arterial stiffness has been suggested to parallel endothelial reactivity and has led researchers to suggest parameters of arterial stiffness may be alternative measures to brachial sonographic assessments of flow-mediated dilatation (FMD). However, past studies comparing these measures can be criticized. In addition to %FMD responses, we recorded concurrent hyperaemic responses of the microcirculation and both were compared with C1 and C2. Twenty-nine subjects 18-30 years of age were investigated. Radial blood pressure was recorded with a tonometer. Pulse waveform analysis was performed to calculate C1 and C2. These were compared with %FMD responses and responses of finger flux measured by laser Doppler fluxmetry (LDF); pulsatile finger volume measured by photoplethysmography (PPG); and palm skin temperature measured by infrared thermography (Tpalm) (i.e. microcirculatory responses). Responses were determined as % changes from control. We only found weak relationships between C1 and %FMD (r=0.4, P=0.04); C2 and %PPG (r=0.38, P=0.07); and C2 and %LDFdorsal (r=-0.38; P=0.04). Responses of %FMD weakly parallel those of C1. Neither C2 nor C1 are viable indicators of endothelial or microcirculatory reactivity (i.e. hyperaemic or venous constriction) in healthy, resting young males. These findings refute the claims that C1 and C2 are substitute measures to sonographic assessments of brachial FMD.

The relation of arterial stiffness to endothelial function in healthy subjects.

Local wall stiffness affects endothelial responsiveness but how global measures affect responsiveness is unanswered. We assessed this by comparing reactive hyperaemic responses of brachial diameter (RHRBD) with central (heart-to-brachial artery pulse wave velocity (PWV); large (C1)) and peripheral (C2) arterial stiffness. Twelve healthy subjects were investigated. RHRBD was induced via an upper- or forearm occluding cuff. Arterial diameter changes were measured using echo ultrasound. Arterial stiffness and RHRBD were compared using a Pearson correlation coefficient (r) and Bland-Altman analysis of Z-scores (indicated as 95% confidence intervals (CI) and expressed in units of standard deviation (SD) from the mean). Weak relations were found between upper-arm RHRBD responses and C2 (r = 0.56, P = 0.06; 95% CI +/- 1.84 SDs) and C1 (r = 0.55, P = 0.06; 95% CI +/- 1.86 SDs). An inverse relation was found between upper-arm RHRBD responses and PWV (r = -0.55, P = 0.06), but Bland-Altman plots revealed no agreement between these parameters (P > 0.05; 95% CI +/- 3.46 SDs). Forearm RHRBD were not related to PWV, C1 or C2 (P > 0.05; 95% CI > 2 SDs). The weak relation between upper-arm endothelial responses and C2 and C1 seems to suggest that C2, and also C1, is not a good and reliable method for assessments of endothelial health. Furthermore, if anything, upper-arm mediated RHRBD responses are more affected by arterial stiffness than forearm responses.

Non-invasive methods and stimuli for evaluating the skin's microcirculation.

Vessels in the skin are arranged into superficial and deep horizontal plexuses and they are involved in thermoregulation, oxygen and nutritional support. The skin has a large number of functions and broad appeal spanning basic mechanistic and clinical research. Indeed, the skin can be used as a marker of normal and impaired vascular control and, owing to its accessibility and frequent involvement, is easy to investigate non-invasively. A large number of non-invasive methods are available for investigating the skin, ranging from those that permit the visualisation of microvessels, to those that monitor blood flow or one of its derivatives (e.g., skin temperature and transcutaneous oxygen). Such methods can be combined with non-invasive, dynamic stimuli (e.g., the use of cold or warm stimuli, activation of the peripheral nervous system or local neuronal systems, and the topical application of vasoactive drugs) and this potentially enables the differentiation of underlying disorders (e.g., primary from secondary Raynaud's phenomenon) and also to quantify changes over time or following intervention. The present article outlines the non-invasive methods and dynamic tests that can be used to investigate the microcirculation of the skin.

Different effects of conjugated linoleic acid isomers on lipoprotein lipase activity in 3T3-L1 adipocytes.

Conjugated linoleic acids (CLAs) are the positional and geometric isomers of linoleic acid. In the present study the effects of cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA ) on intracellular and heparin-releasable (HR-) lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes were investigated. Cells were exposed to the two CLA isomers and linoleic acid, which were bound to bovine serum albumin (BSA). In the adipocytes insulin up-regulated and tumor necrosis factor alpha (TNFalpha) down-regulated HR-LPL activity, which corresponds with the findings in vivo. The experimental fatty acids at low concentrations (<30 µmol/L) moderately increased intracellular and HR-LPL activity. At a concentration of 100 µmol/L, c9,t11 CLA and t10,c12 CLA suppressed HR-LPL activity to 20 and 24% below the BSA control level, respectively, while linoleic acid had no effect unless its concentration was as high as 1000 µmol/L. Insulin abolished the inhibitory effect of c9,t11 CLA, but not of t10,c12 CLA. In the presence of insulin, t10,c12 CLA inhibited HR-LPL activity by 41% compared to BSA control. In contrast to TNFalpha, which suppressed both intracellular LPL and HR-LPL activity, CLAs suppressed HR-LPL activity without decreasing intracellular LPL activity. Additionally, t10,c12 CLA (100 µmol/L) partially prevented TNFalpha-induced decrease of intracellular LPL activity. These results indicate that CLAs differ from linoleic acid in regulating HR-LPL activity, and t10,c12 CLA appeared to be more effective than c9,t11 CLA.

Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: role of protein kinases, calcium, and RhoA.

In the present study, we differentiated between short- and long-term effects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (<3 minutes) decrease in barrier function, as evidenced by a decreased transendothelial electrical resistance and an increased passage of 22Na ions. This increase in permeability was inhibited completely by chelation of intracellular calcium ions by BAPTA-AM and inhibition of calmodulin activity and myosin light chain (MLC) phosphorylation. The presence of serum factors prolonged the barrier dysfunction induced by histamine. Thrombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increase in permeability induced by thrombin. Genistein and BAPTA-AM inhibited the thrombin-induced permeability in an additive way, causing together an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F-actin fiber and focal attachment formation. Inhibition of RhoA by C3 transferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not elevate the cellular cAMP levels. No evidence was found for a significant role of protein kinase C in the thrombin-induced increase in permeability or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a physiological way, thrombin induces a prolonged increase in permeability by "calcium sensitization," which involves protein tyrosine phosphorylation and RhoA activation.

Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin.

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

cGMP and nitric oxide modulate thrombin-induced endothelial permeability. Regulation via different pathways in human aortic and umbilical vein endothelial cells.

Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined whether nitric oxide (NO) can serve as an endogenous modulator of endothelial barrier function. Thrombin increased the passage of macromolecules through human umbilical vein and human aortic endothelial cell monolayers and concomitantly increased [Ca]2+ in vitro. Inhibition of these increases by the intracellular Ca2+ chelator BAPTA indicated that cytoplasmic Ca2+ elevation contributes to the thrombin-induced increase in endothelial permeability. The cGMP-dependent protein kinase activators 8-bromo-cGMP (8-Br-cGMP) and 8-(4-chlorophenylthio)cGMP (8-PCPT-cGMP) decreased the thrombin-induced passage of macromolecules. Two pathways accounted for this observation. Activation of cGMP-dependent protein kinase by 8-PCPT-cGMP decreased the accumulation of cytoplasmic Ca2+ in aortic endothelial cells and hence reduced the thrombin-induced increase in permeability. On the other hand, in umbilical vein endothelial cells, cGMP-inhibited phosphodiesterase (PDE III) activity was mainly responsible for the cGMP-dependent reduction of endothelial permeability. The PDE III inhibitors Indolidan (LY195115) and SKF94120 decreased the thrombin-induced increase in permeability by 50% in these cells. Thrombin treatment increased cGMP formation in the majority of, but not all, cell cultures. Inhibition of NO production by NG-nitro-L-arginine methyl ester (L-NAME) enhanced the thrombin-induced increase in permeability, which was restricted to those cell cultures that displayed an increased cGMP formation after addition of thrombin. Simultaneous elevation of the endothelial cGMP concentration by atrial natriuretic factor, sodium nitroprusside, or 8-Br-cGMP prevented the additional increase in permeability induced by L-NAME. These data indicate that cGMP reduces thrombin-induced endothelial permeability by inhibition of the thrombin-induced Ca2+ accumulation and/or by inhibition of cAMP degradation by PDE III. The relative contribution of these mechanisms differs in aortic and umbilical vein endothelial cells. NO can act in vitro as an endogenous permeability-counteracting agent by raising cGMP in endothelial cells of large vessels.

Cyclic-GMP-mediated decrease in permeability of human umbilical and pulmonary artery endothelial cell monolayers.

Endothelial cell contraction plays a pivotal role in the increased extravasation of fluid and macromolecules in vascular leakage. Previous studies have indicated that elevation of the adenosine 3',5'-cyclic monophosphate (cAMP) concentration can improve the endothelial barrier function. In analogy with smooth muscle cell contraction, which is inhibited by both cAMP and guanosine 3',5'-cyclic monophosphate (cGMP), we have compared the role of cAMP and cGMP in the regulation of the permeability of human endothelial cell monolayers. The cellular cGMP concentration was elevated 3- to 5-fold after addition of 10(-7) M atrial natriuretic peptide (ANP) or 10(-4) M sodium nitroprusside (SNP), both under basal and thrombin-stimulated conditions. After exposure to thrombin, cGMP generation by ANP or SNP or addition of 8-bromo-cGMP significantly suppressed the increase in permeability. Inhibition of nitric oxide production with 10(-4) M NG-nitro-L-arginine methyl ester increased the permeability of endothelial monolayers in the majority of the tested cultures, an effect that could be counteracted by addition of 8-bromo-cGMP or ANP. An increase of cAMP upon the addition of forskolin reduced the permeability in all endothelial cell strains under basal conditions and after exposure to thrombin. The forskolin- and 8-bromo-cGMP-mediated decreases in permeability were accompanied by increases in transendothelial electrical resistance. These in vitro data indicate that, in addition to cAMP, cGMP can act as a potent fine-regulator of endothelial permeability.

Developmental regulation of the inositol 1,4,5-trisphosphate phosphatases in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is a microorganism in which growth and development are strictly separated. Starvation initiates a developmental program in which extracellular cAMP plays a major role as a signal molecule. In response to cAMP several second messengers are produced, including cAMP, cGMP and inositol 1,4,5-trisphosphate, (Ins(1,4,5)P3). Ins(1,4,5)P3 levels are controlled by the activation of phosphoinositidase C and the activity of the Ins(1,4,5)P3-degrading phosphatases. In Dictyostelium discoideum two major routes for the dephosphorylation of Ins(1,4,5)P3 are present: a 5-phosphatase, which hydrolyses Ins(1,4,5)P3 at the 5-position producing Ins(1,4)P2 as in vertebrate cells, and a 1-phosphatase which removes the 1-phosphate, giving Ins(4,5)P2, as in plants. In this paper we show that at the onset of development both the 1-phosphatase and the 5-phosphatase are present in equal amounts. During development the 5-phosphatase disappears leaving the 1-phosphatase as the single enzyme to remove Ins(1,4,5)P3. We conclude that during development Dictyostelium discoideum switches from a mixed type of Ins(1,4,5)P3 degradation to a more plant-like degradation pathway.

Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene.

Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.