Biomedical subjects

Markus van der Giet

Publications and source records attributed to Markus van der Giet.

At least 19 recordsLinked to original sources

PubMed2007

Immunomodulator FTY720 induces myofibroblast differentiation via the lysophospholipid receptor S1P3 and Smad3 signaling.

The novel immunomodulator FTY720 is an effective immunosuppressive agent in experimental models of transplantation and autoimmunity and is currently undergoing phase III clinical trials for multiple sclerosis. Phosphorylated FTY720 is a structural analogue of sphingosine 1-phosphate (S1P) and therefore acts as a high-affinity agonist at four of the five G protein-coupled S1P receptors. It has been well established that there exists a crosstalk between S1P and transforming growth factor (TGF)-beta signaling. Because TGF-beta is the most prominent inductor of fibrosis and myofibroblasts are primarily responsible for excessive matrix protein formation, we examined whether FTY720, in analogy to TGF-beta, induces differentiation of fibroblasts into myofibroblasts. Indeed, FTY720 provoked myofibroblast differentiation comparable with that of TGF-beta. For biological efficacy, FTY720 required endogenous phosphorylation because inhibition of sphingosine kinase completely prevented FTY720 from inducing the differentiation process. Moreover, we identified the lysophospholipid receptor S1P3 as the crucial receptor subtype for FTY720-induced myofibroblast differentiation because the effect was abolished in fibroblasts isolated from S1P3 knockout mice. Finally, we determined that downstream of S1P3 signaling Smad3 activation is essential for myofibroblast differentiation in response to FTY720. Thus, FTY720 may have adverse fibrotic effects related to its activity on S1P3 signaling.

Animals↗

PubMed2006

The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: The sphingosine-1-phosphate (S1P) analogue FTY720 is a potent immunosuppressive agent currently in Phase III clinical trials for kidney transplantation. FTY720 traps lymphocytes in secondary lymphoid organs thereby preventing their migration to inflammatory sites. Previously, we have identified FTY720 as a potent activator of eNOS. As both inhibition of immune responses and stimulation of eNOS may attenuate atherosclerosis, we administered FTY720 to apolipoprotein E-/- mice fed a high-cholesterol diet. METHODS AND RESULTS: FTY720 dramatically reduced atherosclerotic lesion volume (62.5%), macrophage (41.8%), and collagen content (63.5%) after 20 weeks of high-cholesterol diet. In isolated aortic segments and cultured vascular smooth muscle cell, FTY720 potently inhibited thrombin-induced release of monocyte chemoattractant protein-1. This effect was mediated by the S1P3 sphingolipid receptor as FTY720 had no effect on thrombin-induced monocyte chemoattractant protein-1 release in S1P3-/- mice. In contrast to S1P receptors on lymphocytes, FTY720 did not desensitize vascular S1P receptors as arteries from FTY720-treated mice retained their vasodilator response to FTY720-phosphate. CONCLUSIONS: We suggest that FTY720 inhibits atherosclerosis by suppressing the machinery involved in monocyte/macrophage emigration to atherosclerotic lesions. As vascular S1P receptors remained functional under FTY720 treatment, S1P agonists that selectively target the vasculature and not the immune system may be promising new drugs against atherosclerosis.

Animals↗

PubMed2006

Mass-spectrometric identification of a novel angiotensin peptide in human plasma.

OBJECTIVE: Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. METHODS AND RESULTS: Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A/Ang II was higher in end-stage renal failure patients. CONCLUSIONS: Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.

Aged↗

PubMed2006

Time-dependent effects of cadaveric renal transplantation on arterial compliance in patients with end-stage renal disease.

BACKGROUND: Cardiovascular complications are the leading cause of mortality in patients undergoing hemodialysis. Increased arterial stiffness is a strong and independent predictor of cardiovascular risk in these patients. In the present study, we investigated the time-dependent effects of cadaveric renal transplantation on arterial elasticity in end-stage renal disease patients. METHODS: Thirty-six patients underwent successful cadaveric kidney transplantation. Pulse-wave analysis of the radial artery was performed prior to transplantation (day 0) and at six defined intervals after transplantation (day 1 to 90). Compliance of large conduit arteries (C1) and of small resistance arteries (C2) was assessed using a modified Windkessel model of the circulation. RESULTS: Both large artery and small artery compliance were transiently improved within the first four weeks posttransplant reaching a maximum of 122 +/- 42% (C1) and 147 +/- 93% (C2) between day 19 and 23. After the first month, however, elasticity gradually deteriorated to reach baseline values three months after transplantation again. CONCLUSION: The benefits of cadaveric renal transplantation on oscillatory and capacitive artery compliance are only transient. It may be speculated that the subacute and long-term vasoactive effects of calcineurin inhibitors counteract the metabolic benefits of increased renal function on the vasculature.

Adolescent↗

PubMed2006

Diguanosine pentaphosphate: an endogenous activator of Rho-kinase possibly involved in blood pressure regulation.

OBJECTIVE: Rho-kinase activity is increased in cardiovascular disease and in the pathophysiology of hypertension. Few endogenous factors are known that activate the Rho-kinase pathway. Stimulation of P2Y receptors activates the Rho-kinase pathway. Recently identified diguanosine pentaphosphate (Gp5G) possibly activates P2Y receptors. In this study, Gp5G was identified and quantified in human plasma. The influence of Gp5G on vascular tone was studied. METHODS: Gp5G in human plasma was purified to homogeneity by several steps. Gp5G was quantified and identified by matrix-assisted laser desorption/ionization mass spectrometry and enzymatic analysis. The vasoactive effects of Gp5G were studied in the isolated perfused rat kidney and after intra-aortic application. Activation of Rho-kinase was measured using western blot analysis. RESULTS: The plasma level of Gp5G in healthy donors is 9.47 +/- 4.97 nmol/l. Gp5G increases contractile responses induced by angiotensin II in a dose-dependent way [ED50 (-log mol) angiotensin II: 10.9 +/- 0.1; angiotensin II plus Gp5G (100 nmol/l): 11.5 +/- 0.1]. P2 receptor antagonists inhibited the Gp5G-induced increase in angiotensin II vasoconstriction. MRS2179, a selective P2Y1 receptor antagonist, had no effect on Gp5G-mediated angiotensin II potentiation. Rho-kinase inhibition by Y27632 abolished the Gp5G-induced increase of contractile responses to angiotensin II. Concentrations of 10 nmol/l Gp5G activated the translocation of RhoA from the cytosolic to the membranous fraction indicating the activation of Rho-kinase. The intra-aortic application of 100 pmol Gp5G significantly increased mean arterial blood pressure by 13.5 +/- 4.2 mmHg. CONCLUSION: Gp5G is an endogenous activator of Rho-kinase, which might affect vascular tone control by Rho-kinase at physiological levels. Gp5G activates P2Y4&6 receptors, and might play a role in physiological and pathophysiological vascular tone control.

Adult↗

PubMed2005

Altered apolipoprotein A-V expression during the acute phase response is independent of plasma triglyceride levels in mice and humans.

Plasma triglyceride (TG) levels are altered during the acute phase response (APR). Plasma levels of the recently discovered apolipoprotein A-V (apoA-V) are inversely associated with plasma TG. The aim of this study was to investigate the change of apoA-V plasma levels and hepatic apoA-V expression during the APR in relation to plasma TG. During human APR plasma apoA-V was decreased as were plasma TG (each P<0.01). Also early in the course of the murine APR plasma apoA-V levels and hepatic apoA-V expression were decreased and changed in the same direction as plasma TG. Treatment of HepG2 cells with TNF-alpha and IL-1beta decreased apoA-V mRNA levels early by 42% and 55%, respectively (each P<0.001). However, in promoter/reporter assays the human apoA-V promoter was unresponsive to proinflammatory cytokines. Instead, we demonstrate that a significant decrease in apoA-V mRNA stability in response to treatment with TNF-alpha and IL-1beta is the underlying basis of decreased apoA-V expression during the APR (P<0.05). These data demonstrate that (i) apoA-V expression decreases early during the APR due to changes in mRNA stability, and (ii) during the APR apoA-V is not inversely related to plasma TG levels in mice and humans, thereby identifying a relevant pathophysiological setting, in which the previously reported close inverse association between these parameters does not hold true.

Acute-Phase Reaction↗

PubMed2005

GATA4-mediated cardiac hypertrophy induced by d-myo-inositol 1,4,5-tris-phosphate.

We evaluated the effects of d-myo-inositol 1,4,5-tris-phosphate on cardiac hypertrophy. d-myo-inositol 1,4,5-tris-phosphate augmented cardiac hypertrophy as evidenced by its effects on DNA synthesis, protein synthesis, and expression of immediate-early genes c-myc and c-fos, beta-myosin heavy chain, and alpha-actin. The administration of d-myo-inositol 1,4,5-tris-phosphate increased the expression of nuclear factor of activated T-cells and cardiac-restricted zinc finger transcription factor (GATA4). Real-time quantitative RT-PCR showed that d-myo-inositol 1,4,5-tris-phosphate-induced GATA4 mRNA was significantly enhanced even in the presence of the calcineurin inhibitor, cyclosporine A. The effect of d-myo-inositol 1,4,5-tris-phosphate was blocked after inhibition of inositol-trisphosphate receptors but not after inhibition of c-Raf/mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (ERK) or p38 mitogen-activated protein kinase pathways. The study shows that d-myo-inositol 1,4,5-tris-phosphate-induced cardiac hypertrophy is mediated by GATA4 but independent from the calcineurin pathway.

Animals↗

PubMed2005

Arrestin-independent internalization and recycling of the urotensin receptor contribute to long-lasting urotensin II-mediated vasoconstriction.

Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (-log EC50, mol/L:9.0+/-0.1). A second application of UII after 30 minutes elicited a reduced contraction (36+/-4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII ((125)I-UII), approximately 70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t(h)]: 5.6+/-0.2 minutes), but recycled quantitatively within 60 minutes (t(h) 31.9+/-2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction.

Animals↗

PubMed2005

Detection of angiotensin II in supernatants of stimulated mononuclear leukocytes by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass analysis.

Angiotensin II (Ang II) is the major vasoactive component of the renin-angiotensin system. Several components of the renin-angiotensin system have been demonstrated in different tissues. Whereas the roles of tissue and renal renin-angiotensin system have been studied in detail, much less is known on whether the corpuscular elements of circulating blood contribute to Ang II production. Here we examined whether, in addition to vasculature, blood cells also contribute to the circulating Ang II levels. Mononuclear leukocytes were obtained from healthy subjects and were incubated. The resulting supernatant was chromatographed using different chromatographic methods. The vasoconstrictive effects of aliquots of the resulting fractions were tested. Each fraction with a vasoconstrictive effect was analyzed by mass spectrometry. In one fraction with a strong vasoconstrictive effect, Ang II was identified. Mononuclear lymphocytes produced Ang II in amounts sufficient to stimulate Ang II type 1 receptors. Moreover, in mononuclear leukocytes, renin as well as angiotensin-converting enzyme mRNA expression was detectable by RT-PCR. These findings demonstrate that mononuclear leukocytes are a source of Ang II. Ang II secretion by these cells may play a significant role in humoral vascular regulation. In conclusion, the isolation of Ang II in supernatants of mononuclear leukocytes adds a further physiological source of Ang II to the current view of angiotensin metabolism. The quantitative role of lymphocyte-derived Ang II secretion compared with the other sources of Ang II should be defined further, but the release found under the present conditions is at least sufficient to elicit vasoconstrictive effects.

Angiotensin II↗

PubMed2005

A physiogenomic approach to study the regulation of blood pressure.

Several vasoregulatory systems including the renin-angiotensin system, sympathetic vasoregulation, and cytokine release have been studied extensively. The aim of the present study was to establish a physiogenomic screening model for differentially expressed genes in the regulation of blood pressure that might give a hint as to new vasoregulatory mechanisms. We induced acute hypotension in normotensive rats, assuming that vasoregulatory systems will counteract hypotension. Microarray transcriptome analysis was performed from kidneys 6 h after the induction of acute hypotension. The results were confirmed by real-time PCR. Six functionally known genes (Igfbp1, Xdh, Sult1a1, Mawbp, Por, and Gstm1) and two expressed sequence tages (BI277460 and AI411345) were significantly upregulated. Four of these genes (Igfbp1, Xdh, Por, and Gstm1) have well-characterized functions in the cardiovascular system. The proteins corresponding to Xdh, Por, and Gstm1 are involved in the metabolism of reactive oxygen species (ROS). Because ROS can mediate endothelial dysfunction, we measured the aortic dilatory capacity in thoracic aortic rings. Indeed, vasodilator potency to acetylcholine was largely diminished in hypotensive animals, whereas sodium nitroprusside induced equivalent vasodilations in normotensive and hypotensive animals. The vasodilator potency of the endothelium was partially restored by the superoxide scavenger tiron. Hence, acute hypotension induces a diminished vasodilator potency of the endothelium due to an accelerated degradation of nitric oxide by ROS. The present physiogenomic approach is capable of detecting vasoregulatory mechanisms and may provide deeper insight into the genetics and physiology of blood pressure regulation.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2005

Uridine adenosine tetraphosphate: a novel endothelium- derived vasoconstrictive factor.

Beyond serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors (EDCFs) remains unclear. The supernatant obtained from mechanically stimulated human endothelial cells obtained from dermal vessels elicited a vasoconstrictive response in an isolated perfused rat kidney. A purinoceptor blocker had a greater effect than an endothelin receptor blocker in decreasing endothelially derived vasoconstriction in the isolated perfused rat kidney. The nucleotide uridine adenosine tetraphosphate (Up(4)A) was isolated from the supernatant of stimulated human endothelium and identified by mass spectrometry. Up(4)A is likely to exert vasoconstriction predominantly through P2X1 receptors, and probably also through P2Y2 and P2Y4 receptors. Plasma concentrations of Up(4)A that cause vasoconstriction are found in healthy subjects. Stimulation with adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), acetylcholine, endothelin, A23187 and mechanical stress releases Up(4)A from endothelium, suggesting that Up(4)A contributes to vascular autoregulation. To our knowledge, Up(4)A is the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. We conclude that Up(4)A is a novel potent nonpeptidic EDCF. Its vasoactive effects, plasma concentrations and its release upon endothelial stimulation strongly suggest that Up(4)A has a functional vasoregulatory role.

Adenine Nucleotides↗

PubMed2005

Convenience of ambulatory blood pressure monitoring: comparison of different devices.

OBJECTIVE: Twenty-four-hour ambulatory blood pressure monitoring has emerged as an important tool supporting physicians in the diagnosis and management of arterial hypertension. Compared with office measurements and self-measurements, however, ambulatory blood pressure monitoring shows the lowest patients' acceptance. The present study compares the convenience of different monitors in order to examine whether the patients benefit from new technologies. METHODS: In a prospective randomized study, we compared the side effects of the Spacelabs 90207 with the I.E.M. Mobilograph monitor in 250 patients by means of a questionnaire that covered the following aspects: restriction in everyday activities, noise, sleep disturbance, pain and mobility. Complaints were measured by a five-point scale ranging from 'no complaints at all' to 'severe complaints'. RESULTS: In all, 205 patients returned completed surveys (101 patients of the I.E.M. group, 104 patients of the Spacelabs group), yielding an 82% final response rate. The overall mean complaint score was significantly higher in the Spacelabs group than in the I.E.M group (2.24 vs. 1.78; P<0.001). The Mobilograph revealed less discomfort in every single question and differences were significant for restrictions in everyday activities, noise, pain, disturbance of the patient's or the patient's partner's sleep and restrictions in walking. Sleep disturbance was the aspect with the highest difference in the two groups. CONCLUSION: The present work confirms that ambulatory blood pressure monitoring monitors are cumbersome to wear. Devices, however, differ in their comfort. Monitors with an improved convenience might lead to a higher patients' acceptance of this powerful diagnostic tool.

Activities of Daily Living↗

PubMed2004

High-density lipoprotein stimulates myocardial perfusion in vivo.

BACKGROUND: Several clinical studies have demonstrated a close association between plasma HDL cholesterol levels and endothelium-dependent vasodilation in peripheral arteries. In isolated arteries, HDL has been shown to mediate vasodilation via NO release. In vivo, administration of reconstituted HDL restored abnormal endothelial function of the brachial artery in hypercholesterolemic patients. However, no data are currently available on the effect of HDL on myocardial perfusion. METHODS AND RESULTS: In this study, administration of human HDL enhanced incorporation of the perfusion tracer 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) into the murine heart in vivo by approximately 18%. This increase was completely abolished in mice deficient for endothelial NO synthase. Because we have recently identified sphingosine 1-phosphate (S1P) as an important vasoactive component contained in HDL, we measured myocardial perfusion after administration of S1P in vivo. We observed an approximately 25% decrease in myocardial MIBI uptake, which was abolished in mice deficient for the S1P receptor S1P3. In S1P3-/- mice, the stimulatory effect of HDL on myocardial perfusion was preserved. CONCLUSIONS: HDL increased myocardial perfusion under basal conditions in vivo via NO-dependent mechanisms, whereas S1P inhibited myocardial perfusion through the S1P3 receptor. Thus, HDL may reduce coronary risk via direct NO-mediated vasodilatory effects on the coronary circulation.

Animals↗

PubMed2004

Effect of sodium on vasoconstriction and angiotensin II type 1 receptor mRNA expression in cold-induced hypertensive rats.

Angiotensin II and sodium play an important pathogenetic role in several models of hypertension. Now, we investigated the effects of sodium on vasoconstriction and angiotensin II type 1 (AT1) and type 2 (AT2) receptor mRNA expression in aortic vessels from cold-induced hypertensive rats. Wistar rats on low sodium and high sodium diet were exposed to cold-stress for 8 weeks. The effects of angiotensin II infusion on mean arterial blood pressure were investigated in these rats. In addition, angiotensin II induced contraction was measured using aortic rings. Expression of AT1 receptor mRNA and AT2 receptor mRNA was assessed in aortic vessels by reverse transcription polymerase chain reaction. After infusion of angiotensin II mean arterial blood pressure in cold-induced hypertensive rats on high sodium diet was significantly higher compared to cold-induced hypertensive rats on low sodium diet (p < 0.05). Angiotensin II-induced contraction of aortic rings was significantly higher in cold-induced hypertensive rats on high sodium diet compared to cold-induced hypertensive rats on low sodium diet (2.39 +/- 0.03 g vs. 2.21 +/- 0.04 g, n = 12, p < 0.01). Angiotensin AT1 receptor mRNA was significantly higher in cold-induced hypertensive rats on high sodium diet compared to cold-induced hypertensive rats on low sodium diet (p < 0.05). It is concluded that in this nongenetic, nonsurgical animal model of cold-induced hypertension increased vasoconstriction and increased AT1 receptor mRNA expression in aortic vessels are dependent on sodium intake.

Animals↗

PubMed2004

HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3.

HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.

Animals↗

PubMed2003

Identification and quantification of diadenosine polyphosphate concentrations in human plasma.

OBJECTIVE: Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasma and whether a potential source can be identified that may release diadenosine polyphosphates into the circulation. METHODS AND RESULTS: Plasma diadenosine polyphosphates (ApnA with n=3 to 6) were purified to homogeneity by affinity-, anion exchange-, and reversed phase-chromatography from deproteinized human plasma. Analysis of the homogeneous fractions with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed molecular masses ([M+H]+) of 757, 837, 917, and 997 d. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrometry mass spectra of these fractions with those of authentic diadenosine polyphosphates revealed that these isolated substances were identical to Ap3A, Ap4A, Ap5A, and Ap6A. Enzymatic analysis showed an interconnection of the phosphate groups with the adenosines in the 5'-positions of the ribose moieties. The mean total plasma diadenosine polyphosphate concentrations (micromol/L; mean +/- SEM) in cubital veins of normotensive subjects amounted to 0.89+/-0.59 for Ap3A, 0.72+/-0.72 for Ap4A, 0.33+/-0.24 for Ap5A, and 0.18+/-0.18 for Ap6A. Cubital venous plasma diadenosine polyphosphate concentrations from normotensives did not differ significantly from those in the hypertensive patients studied. There was no significant difference between arterial and venous diadenosine polyphosphate plasma concentrations in 5 hemodialysis patients, making a significant degradation by capillary endothelial cells unlikely. Free plasma diadenosine polyphosphate concentrations are considerably lower than total plasma concentrations because approximately 95% of the plasma diadenosine polyphosphates were bound to proteins. There were no significant differences in the diadenosine polyphosphate plasma concentrations depending on the method of blood sampling and anticoagulation, suggesting that platelet aggregation does not artificially contribute to plasma diadenosine polyphosphate levels in significant amounts. The ApnA (with n=3 to 6) total plasma concentrations in adrenal veins were significantly higher than the plasma concentrations in both infrarenal and suprarenal vena cava: adrenal veins: Ap3A, 4.05+/-1.63; Ap4A, 6.18+/-2.08; Ap5A, 0.53+/-0.28; Ap6A, 0.59+/-0.31; infrarenal vena cava: Ap3A, 1.25+/-0.66; Ap4A, 0.91+/-0.54; Ap5A, 0.25+/-0.12; Ap6A, 0.11+/-0.06; suprarenal vena cava: Ap3A, 1.40+/-0.91; Ap4A, 1.84+/-1.20; Ap5A, 0.33+/-0.13; Ap6A, 0.11+/-0.07 (micromol/L; mean +/- SEM; each P<0.05 (concentration of adrenal veins versus infrarenal or suprarenal veins, respectively). CONCLUSIONS: The presence of diadenosine polyphosphates in physiologically relevant concentrations in human plasma was demonstrated. Because in adrenal venous plasma significantly higher diadenosine polyphosphate concentrations were measured than in plasma from the infrarenal and suprarenal vena cava, it can be assumed that, beside platelets, the adrenal medulla may be a source of plasma diadenosine polyphosphates in humans.

Chromatography, High Pressure Liquid↗

PubMed2003

Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue.

Endocrine functions of the human heart have been studied extensively. Only recently, nucleotidergic mechanisms have been studied in detail. Therefore, an isolation strategy was developed to isolate novel nucleotide compounds from human myocardium. The human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5'-tetraphosphate (Ap(4)) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P(2Y1) receptor antagonist MRS 2179 (1 microm) or the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (50 microm). After removal of the endothelium by Triton X-100, Ap(4) induced dose-dependent vasoconstrictions. Inhibition of P(2X) receptors by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 microm) or desensitization of P(2X) receptors by alpha,beta-methylene ATP (alpha,beta-meATP, 1 microm) diminished these vasoconstrictor responses completely. In the present study Ap(4) has been isolated from human tissue. Ap(4) was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P(2Y1) receptors and vasoconstriction via P(2X) receptors on vascular smooth muscle cells. Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.

Adenine Nucleotides↗

PubMed2003

The antioxidant acetylcysteine reduces cardiovascular events in patients with end-stage renal failure: a randomized, controlled trial.

BACKGROUND: Patients with end-stage renal failure have increased oxidative stress and show elevated cardiovascular mortality. Whether increased cardiovascular events can be prevented by the administration of antioxidants is unknown. METHODS AND RESULTS: We evaluated the effects of acetylcysteine, a thiol-containing antioxidant, on cardiovascular events in patients undergoing hemodialysis. A prospective, randomized, placebo-controlled trial was conducted between October 1, 1999, and September 30, 2001, in 134 patients (76 male and 58 female) with a mean age of 62+/-16 years (mean+/-SD) who had been undergoing maintenance hemodialysis for a minimum of 3 months 3 times weekly in an ambulatory center. Median (range) follow-up was 14.5 (1 to 24) months. Patients were randomly assigned either to receive acetylcysteine (600 mg BID) or placebo. The primary end point was a composite variable consisting of cardiac events including fatal and nonfatal myocardial infarction, cardiovascular disease death, need for coronary angioplasty or coronary bypass surgery, ischemic stroke, peripheral vascular disease with amputation, or need for angioplasty. Secondary end points included each of the component outcomes, total mortality, and cardiovascular mortality. A total of 18 (28%) of the 64 hemodialysis patients assigned to acetylcysteine group and 33 (47%) of the 70 hemodialysis patients assigned to control group had a primary end point (relative risk, 0.60 [95% CI, 0.38 to 0.95], P=0.03). No significant differences in secondary end points or total mortality were detected. CONCLUSIONS: In hemodialysis patients, treatment with acetylcysteine (600 mg BID) reduces composite cardiovascular end points.

Acetylcysteine↗