Lars Henning

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 (Up4A) was isolated from the supernatant of stimulated human endothelium and identified by mass spectrometry. Up4A is likely to exert vasoconstriction predominantly through P2X1 receptors, and probably also through P2Y2 and P2Y4 receptors. Plasma concentrations of Up4A 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 Up4A from endothelium, suggesting that Up4A contributes to vascular autoregulation. To our knowledge, Up4A is the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. We conclude that Up4A is a novel potent nonpeptidic EDCF. Its vasoactive effects, plasma concentrations and its release upon endothelial stimulation strongly suggest that Up4A has a functional vasoregulatory role.

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 (&mgr;mol/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 (&mgr;mol/L; mean ± SEM; each P <0.05 (concentration of adrenal veins versus infrarenal or suprarenal veins, respectively). Conclusion—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.

Dinucleotides as Growth-promoting Extracellular Mediators PRESENCE OF DINUCLEOSIDE DIPHOSPHATES Ap2A, Ap2G, AND Gp2G IN RELEASABLE GRANULES OF PLATELETS

Dinucleoside diphosphates, Ap2A, Ap2G, and Gp2G represent a new class of growth-promoting extracellular mediators, which are released from granules after activation of platelets. The presence of theses substances was shown after purification from a platelet concentrate. The substances were identified by UV spectrometry, retention time comparison with authentic substances, matrix-assisted laser desorption/ionization mass spectrometry, post-source-decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic analysis. Ap2A, Ap2G, and Gp2G have growth-stimulating effects on vascular smooth muscle cells in nanomolar concentrations as shown by [3H]thymidine incorporation measurements. The calculated EC50 (log m; mean ± S.E.) values were −6.07 ± 0.14 for Ap2A, −6.27 ± 0.25 for Ap2G, and −6.91 ± 0.44 for Gp2G. At least 61.5 ± 4.3% of the dinucleoside polyphosphates are released by platelet activation. The intraplatelet concentrations suggest that, in the close environment of a platelet thrombus, similar dinucleoside polyphosphate concentrations can be found as in platelets. Intraplatelet concentration can be estimated in the range of 1/20 to 1/100 of the concentration of ATP. In conclusion, Ap2A, Ap2G, and Gp2G derived from releasable granules of human platelets may play a regulatory role in vascular smooth muscle growth as growth-promoting mediators.

Detection of Angiotensin II in Supernatants of Stimulated Mononuclear Leukocytes by Matrix-Assisted Laser Desorption Ionization Time-of-Flight/Time-of-Flight Mass Analysis

Abstract—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.

Phenylacetic acid and arterial vascular properties in patients with chronic kidney disease stage 5 on hemodialysis therapy.

Background: Phenylacetic acid (PAA) is a recently described uremic toxin that inhibits inducible nitric oxide synthase expression and plasma membrane calcium ATPase and may therefore also be involved in remodeling of arteries. Such vascular effects have not been evaluated yet in patients with chronic kidney disease stage 5. Method: We prospectively measured the plasma concentrations of PAA using nuclear magnetic resonance spectroscopy in 50 patients with chronic kidney disease stage 5 (37 men, 13 women) on maintenance hemodialysis. Arterial vascular properties were quantified by the reflective index obtained from digital photoplethysmography. Results: During the hemodialysis session the plasma PAA concentration was reduced from 3.38 ± 0.24 mmol/l (mean ± SEM; median, 2.85 mmol/l; interquartile range, 2.02–4.52 mmol/l) to 2.25 ± 0.11 mmol/l (median, 2.06 mmol/l; interquartile range, 1.62–2.86 mmol/l; n = 50; p < 0.001). There was a significant correlation between the PAA concentration and the reflective index before the start of the hemodialysis session. Conclusion: The study demonstrates an association of PAA and arterial vascular properties in patients with chronic kidney disease stage 5.

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.

The AN69 hemofiltration membrane has a decreasing effect on the intracellular diadenosine pentaphosphate concentration of platelets

Background/Aim: The biocompatibility of hemodialysis membranes has a substantial impact on the mortality of patients with end-stage renal failure. In the present study, the effects of hemodialysis on the intracellular amount of diadenosine pentaphosphate (Ap5A), a hydrophilic, anionic substance with a low molecular weight, was investigated. Methods: The intracellular Ap5A concentrations were measured before and after hemodialysis using either polyacrylonitrile (AN69; n = 10) or polysulfone (n = 23) membranes. Ap5A was isolated from platelets using affinity chromatography and reversed-phase chromatography methods. Results: The Ap5A concentrations were quantified by ultraviolet absorption at 254 nm. The Ap5A concentrations were significantly higher in platelets from the patients with end-stage renal failure as compared with the 21 healthy control subjects (136 ± 50 vs. 9 ± 6 fg/platelet; mean ± SEM, p < 0.01). Before hemodialysis, the intracellular Ap5A concentrations in platelets from 10 patients with end-stage renal failure using an AN69 membrane were not significantly different from those in platelets from 23 patients using a polysulfone membrane (93 ± 39 vs. 155 ± 70 fg/platelet). However, after a hemodialysis session, the intracellular Ap5A concentrations in platelets from patients with end-stage renal failure using an AN69 membrane were significantly lower as compared with those in platelets before hemodialysis (51 ± 18 vs. 93 ± 39 fg/platelet, p < 0.05) as well as compared with those in platelets from patients using a polysulfone membrane (51 ± 18 vs. 250 ± 59 fg/platelet, p < 0.05). Conclusions: It was found that hemofiltration by using an AN69 membrane has a direct effect on the intracellular amount of Ap5A and that changes of intracellular hydrophilic substances are dependent on the hemodialysis membrane used.

Endogenous coenzyme A glutathione disulfide in human myocardial tissue

Besides its role as a mechanical pump, the human heart serves as an endocrine organ, where known and as yet unknown hormones are produced. It is very likely that these hormones play an important role in cardiovascular regulation. In this study, a new endogenous vasoactive substance, coenzyme A glutathione disulfide (CoASSG), was isolated and identified in myocardial tissue. Human myocardial tissue was extracted with perchloric acid and fractionated by size exclusion-, displacement-, anion-exchange- and reversed-phase chromatography. In one fraction purified to homogeneity, CoASSG was identified by matrix assisted laser desorption/ionization (MALDI) mass-spectrometry, post-source decay MALDI-massspectrometry and enzymatic structure analysis. Furthermore, CoASSG was also isolated from human cardiac specific granules. CoASSG has potent vasoconstrictive and proliferative effects. Therefore, CoASSG may affect myocardial function as an endocrine or autocrine substance after being released from myocardial specific granules.