Vasodilator effects of L-arginine are stereospecific and augmented by insulin in humans

The amino acid l-arginine, the precursor of nitric oxide (NO) synthesis, induces vasodilation in vivo, but the mechanism behind this effect is unclear. There is, however, some evidence to assume that the l-arginine membrane transport capacity is dependent on insulin plasma levels. We hypothesized that vasodilator effects of l-arginine may be dependent on insulin plasma levels. Accordingly, we performed two randomized, double-blind crossover studies in healthy male subjects. In protocol 1 (n = 15), subjects received an infusion of insulin (6 mU x kg(-1) x min(-1) for 120 min) or placebo and, during the last 30 min, l-arginine or d-arginine (1 g/min for 30 min) x In protocol 2 (n = 8), subjects received l-arginine in stepwise increasing doses in the presence (1.5 mU x kg(-1) x min(-1)) or absence of insulin. Renal plasma flow and glomerular filtration rate were assessed by the para-aminohippurate and inulin plasma clearance methods, respectively. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation, and mean flow velocity in the ophthalmic artery was measured with Doppler sonography. l-arginine, but not d-arginine, significantly increased renal and ocular hemodynamic parameters. Coinfusion of l-arginine with insulin caused a dose-dependent leftward shift of the vasodilator effect of l-arginine. This stereospecific renal and ocular vasodilator potency of l-arginine is enhanced by insulin, which may result from facilitated l-arginine membrane transport, enhanced intracellular NO formation, or increased NO bioavailability.     

Hypercapnia-induced cerebral and ocular vasodilation is not altered by glibenclamide in humans

Carbon dioxide is an important regulator of vascular tone. Glibenclamide, an inhibitor of ATP-sensitive potassium channel (K(ATP)) activation, significantly blunts vasodilation in response to hypercapnic acidosis in animals. We investigated whether glibenclamide also alters the cerebral and ocular vasodilator response to hypercapnia in humans. Ten healthy male subjects were studied in a controlled, randomized, double-blind two-way crossover study under normoxic and hypercapnic conditions. Glibenclamide (5 mg po) or insulin (0.3 mU. kg(-1). min(-1) iv) were administered with glucose to achieve comparable plasma insulin levels. In control experiments, five healthy volunteers received glibenclamide (5 mg) or nicorandil (40 mg) or glibenclamide and nicorandil in a randomized, three-way crossover study. Mean blood flow velocity and resistive index in the middle cerebral artery (MCA) and in the ophthalmic artery (OA) were measured with Doppler sonography. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation. Forearm blood flow was measured with venous occlusion plethysmography. Hypercapnia increased ocular fundus pulsation amplitude by +18.2-22.3% (P < 0. 001) and mean flow velocity in the MCA by +27.4-33.3% (P < 0.001), but not in the OA (2.1-6.5%, P = 0.2). Forearm blood flow increased by 78.2% vs. baseline (P = 0.041) after nicorandil administration. Glibenclamide did not alter hypercapnia-induced changes in cerebral or ocular hemodynamics and did not affect systemic hemodynamics or forearm blood flow but significantly increased glucose utilization and blunted the nicorandil-induced vasodilation in the forearm. This suggests that hypercapnia-induced changes in the vascular beds under study are not mediated by activation of K(ATP) channels in humans.     

Optic Nerve Head Blood Flow Autoregulation during Changes in Arterial Blood Pressure in Healthy Young Subjects

Aim

In the present study the response of optic nerve head blood flow to an increase in ocular perfusion pressure during isometric exercise was studied. Based on our previous studies we hypothesized that subjects with an abnormal blood flow response, defined as a decrease in blood flow of more than 10% during or after isometric exercise, could be identified.

Methods

A total of 40 healthy subjects were included in this study. Three periods of isometric exercise were scheduled, each consisting of 2 minutes of handgripping. Optic nerve head blood flow was measured continuously before, during and after handgripping using laser Doppler flowmetry. Blood pressure was measured non-invasively in one-minute intervals. Intraocular pressure was measured at the beginning and the end of the measurements and ocular perfusion pressure was calculated as 2/3*mean arterial pressure –intraocular pressure.

Results

Isometric exercise was associated with an increase in ocular perfusion pressure during all handgripping periods (p < 0.001). By contrast no change in optic nerve head blood flow was seen. However, in a subgroup of three subjects blood flow showed a consistent decrease of more than 10% during isometric exercise although their blood pressure values increased. In addition, three other subjects showed a consistent decline of blood flow of more than 10% during the recovery periods.

Conclusion

Our data confirm previous results indicating that optic nerve head blood flow is autoregulated during an increase in perfusion pressure. In addition, we observed a subgroup of 6 subjects (15%) that showed an abnormal response, which is in keeping with our previous data. The mechanisms underlying this abnormal response remain to be shown.     

General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines

Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcgamma receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 x 10(4) units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future.     

The renal and systemic hemodynamic effects of a nitric oxide-synthase inhibitor are reversed by a selective endothelin(a) receptor antagonist in men.

There is evidence for an interaction between nitric oxide (NO) and endothelin (ET) at the level of the renal vasculature. We hypothesized that acute renal effects of systemic NO synthase inhibition (NG-monomethyl-l-arginine, L-NMMA) may be blunted by coadministration of a specific ET(A) receptor antagonist (BQ-123) in healthy humans. Fifteen healthy young male subjects participated in this randomized, double-blind, placebo-controlled 3-way crossover study. These sodium-repleted volunteers received L-NMMA alone, or BQ-123 alone, or L-NMMA with a subsequent coinfusion of BQ-123. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were determined with the PAH and inulin clearance method, respectively. Mean arterial pressure (MAP) and pulse rate were measured noninvasively at baseline and every 15 min after the start of the study period. L-NMMA alone reduced RPF (-22%, P < 0.001) and GFR (-8%, P < 0.009) and increased MAP (+10%, P < 0.001). BQ-123 alone did not affect these parameters. However, coinfusion of BQ-123 blunted the effects of L-NMMA on RPF (P < 0.001), GFR (P < 0.001), and MAP (P = 0.006). Peripheral and renal hemodynamic effects of acute systemic NO synthase inhibition are at least partially reversed by ET(A) receptor blockade with BQ-123. This indicates a functional antagonism between specific ET(A) receptor antagonist and NO synthase inhibitors at the level of the renal vasculature.     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.     

Oxidation of exogenous c-type cytochromes by intact spheroplasts of Anacystis nidulans

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize reduced c-type cytochromes derived from horse heart, tuna, Saccharomyces oviformis, Candida krusei, Rhodocyclus purpureus, Rhodopseudomonas plustris and Paracoccus denitrificans with characteristics similar to those observed with isolated membranes. Rates of cytochrome c oxidation by the spheroplasts were only 10% of those measured with isolated membranes in which thylakoid-bound cytochrome oxidase contributes to the overall rates. Small amounts of an endogenous c-type cytochrome were released upon lysozyme treatment of the cells. The results appear to indicate the presence of cytochrome oxidase in the cytoplasmic membrane of A. nidulans.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - cyt cytochrome(s)     

LPS-induced microvascular leukocytosis can be assessed by blue-field entoptic phenomenon

Administration of low doses of Escherichia coli endotoxin [a lipopolysaccharide (LPS)] to humans enables the study of inflammatory mechanisms. The purpose of the present study was to investigate whether the blue-field entoptic technique may be used to quantify the increase in circulating leukocytes in the ocular microvasculature after LPS infusion. In addition, combined laser Doppler velocimetry and retinal vessel size measurement were used to study red blood cell movement. Twelve healthy male volunteers received 20 IU/kg iv LPS as a bolus infusion. Outcome parameters were measured at baseline and 4 h after LPS administration. In the first protocol (n = 6 subjects), ocular hemodynamic effects were assessed with the blue-field entoptic technique, the retinal vessel analyzer, and laser Doppler velocimetry. In the second protocol (n = 6 subjects), white blood cell (WBC) counts from peripheral blood samples and blue-field entoptic technique measurements were performed. LPS caused peripheral blood leukocytosis and increased WBC density in ocular microvessels (by 49%; P = 0.036) but did not change WBC velocity. In addition, retinal venous diameter was increased (by 9%; P = 0.008), but red blood cell velocity remained unchanged. The LPS-induced changes in retinal WBC density and leukocyte counts were significantly correlated (r = 0.87). The present study indicates that the blue-field entoptic technique can be used to assess microvascular leukocyte recruitment in vivo. In addition, our data indicate retinal venous dilation in response to endotoxin.