Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 lead to downstream activation of eNOS in HUVEC in the same experimental setting. Inhibition or knockdown of one of these receptor subtypes did not abolish the eNOS activation and subsequent NO production.

Guanidinylations of albumin decreased binding capacity of hydrophobic metabolites.

AIM: As post-translational modifications of proteins may have an impact on the pathogenesis of diseases such as atherosclerosis, diabetes mellitus and chronic kidney disease (CKD), post-translational modifications are currently gaining increasing interest. In this study, a comprehensive method for analysis of these post-translational modifications is established for the clinical diagnostic routine. METHODS: Here, we analysed albumin - the most abundant plasma protein in human - isolated from patients with CKD and healthy controls by chromatographic steps and identified by MALDI mass spectrometry. Post-translational modifications of albumin were identified after digestion by analysing mass signal shifts of albumin peptides using pertinent mass databases. RESULTS: Albumin isolated from plasma of patients with CKD but not from healthy control subjects was specifically post-translationally modified by guanidinylation of lysines at positions 249, 468, 548, 565 and 588. After identification of guanidinylations as post-translational modifications of albumin isolated from patients with CKD, these modifications were quantified by mass spectrometry demonstrating a significant increase in the corresponding mass signal intensities in patients with CKD compared to healthy controls. The relative amount of guanidinylation of lysine at position 468 in patients with CKD was determined as 63 ± 32% (N = 3). Subsequently, we characterized the pathophysiological impact of the post-translational guanidinylation on the binding capacity of albumin for representative hydrophobic metabolic waste products. In vitro guanidinylation of albumin from healthy control subjects caused a decreased binding capacity of albumin in a time-dependent manner. Binding of indoxyl sulphate (protein-bound fraction) decreased from 82 ± 1% of not post-translationally modified albumin to 56 ± 1% after in vitro guanidinylation (P < 0.01), whereas the binding of tryptophan decreased from 20 to 4%. These results are in accordance with the binding of indoxyl sulphate to albumin from healthy control subjects and patients with CKD (88 ± 3 vs. 74 ± 10, P < 0.01). Thus, in vitro post-translational guanidinylation of albumin had a direct effect on the binding capacity of hydrophobic metabolites such as indoxyl sulphate and tryptophan. CONCLUSION: We used a mass spectrometry-based method for the characterization of post-translational modification and demonstrated the pathophysiological impact of a representative post-translational modification of plasma albumin. The data described in this study may help to elucidate the pathophysiological role of protein modifications.

Renal denervation for refractory hypertension - technical aspects, complications and radiation exposure.

PURPOSE: To analyze procedural details, complications and radiation exposure in renal denervation (RDN) using the Medtronic Symplicity® device in the treatment of refractory hypertension. MATERIALS AND METHODS: Fifty three consecutive patients underwent RDN. The number of ablations per artery, peri-procedural complications, procedure time (PT), fluoroscopy time (FT), dose-area product (DAP) and procedure-related complications were documented. Additionally, the radiation dose was compared between obese (body mass index ≥ 30 kg/m(2)) and non-obese patients. RESULTS: Bilateral RDN was performed in 50/53 (94 %) cases and with a minimum of 4 ablations per artery in 33/50 (66 %), the mean count being 5.4 (range R: 2 - 13) on the right and 4.3 (R: 1 - 10) on the left. The FT and DAP decreased significantly over the first 12 procedures, reaching a steady state with a median FT of 11.2 min (R: 7.5 - 27) and a median DAP of 4796 cGy × cm(2) (R: 1076 - 21 371), resulting in an effective dose of 15.7 mSv. The median PT was 57 min (R: 40 - 70). Obese patients had a 3.3-fold higher radiation dose (p < 0.001). We observed one severe spasm and one imminent respiratory depression, both resolved without sequelae. CONCLUSION: For an experienced interventionalist, RDN has a short learning curve with a low risk profile. The radiation dose does not exceed that of other renal artery interventions, but is explicitly higher in obese patients, who account for a large portion of patients with refractory hypertension.

Calprotectin and neutrophil gelatinase-associated lipocalin in the differentiation of pre-renal and intrinsic acute kidney injury.

BACKGROUND: Urinary calprotectin has recently been identified as a promising biomarker for the differentiation of pre-renal and intrinsic acute kidney injury (AKI). This study compares the diagnostic performance of calprotectin and neutrophil gelatinase-associated lipocalin (NGAL) in this differential diagnosis. METHODS: Urinary calprotectin and NGAL concentrations were assessed in a study population of 87 subjects including 38 cases of intrinsic AKI, 24 cases of pre-renal AKI and 25 healthy controls. Urinary tract obstruction, renal transplantation and metastatic cancer were defined as exclusion criteria. RESULTS: Mean calprotectin concentrations were significantly lower in pre-renal (190.2 ± 205.7 ng mL(-1) ) than in intrinsic AKI (6250.1 ± 7167.2 ng mL(-1) , P < 0.001). Receiver-operating characteristic (ROC) analysis provided an AUC of 0.99. Mean NGAL concentrations were significantly higher in intrinsic than in pre-renal AKI as well (458.1 ± 695.3 vs. 64.8 ± 62.1 ng mL(-1) , P = 0.001) providing an AUC of 0.82. A combination of the present study population with the cohort of the proof of concept study led to a population of 188 subjects (58 pre-renal AKI, 90 intrinsic AKI, 40 healthy controls). ROC analyses provided an AUC of 0.97 for calprotectin and 0.76 for NGAL yielding sensitivity and specificity values of 93.3 and 94.8% (calprotectin) vs. 75.3 and 72.4% (NGAL). Optimal cut-off values were 440 ng mL(-1) (calprotectin) and 52 ng mL(-1) (NGAL). Pyuria increased calprotectin concentrations independent of renal failure. CONCLUSION: This study shows that both calprotectin and NGAL are able to differentiate between pre-renal and intrinsic AKI after exclusion of pyuria. In the present population, calprotectin presents a higher sensitivity and specificity than NGAL.

Replacement of acetonitrile by ethanol as solvent in reversed phase chromatography of biomolecules.

Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water-acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules.

Uridine adenosine tetraphosphate acts as an autocrine hormone affecting glomerular filtration rate.

Recently, uridine adenosine tetraphosphate (Up(4)A) was described as a strong vasoconstrictor released from endothelial cells after stimulation with mechanical stress. In this study, we isolated and identified Up(4)A from kidney tissue, and we characterized the essential varying effects of Up(4)A on the afferent and efferent arterioles. Porcine and human kidney tissue was fractionated by size exclusion chromatography, affinity chromatography, anion exchange chromatography and reverse phase chromatography. In fractions purified to homogeneity, Up(4)A was identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), MALDI-LIFT fragment mass spectrometry (MALDI-TOF/TOF MS), retention-time comparison and enzymatic cleavage analysis. We analysed the release of Up(4)A from cultivated renal proximal tubule cells after stimulation of protein kinase C with oleoyl-2-acetyl-sn-glycerol (OAG). Up(4)A was identified in renal tissue, and the effect of Up(4)A on the vascular tone of isolated perfused afferent and efferent arterioles was tested. Stimulation of tubule cells with OAG increased the release rate of Up(4)A from tubule cells about tenfold. Up(4)A acts as a strong vasoconstrictive mediator on afferent arterioles, but has no significant effect on the tone of efferent arterioles, suggesting a functional role of Up(4)A as an autocrine hormone for glomerular perfusion. Because of the predominant effect of the Up(4)A on afferent arterioles, we assume that Up(4)A may decrease glomerular perfusion, intra-glomerular pressure and, hence, glomerular filtration rate. The release of Up(4)A from renal tubular cells may be an additional mechanism whereby tubular cells could affect renal perfusion. Up(4)A release may further contribute to renal vascular autoregulation mechanisms. In conclusion, as Up(4)A occurs in renal tissue and has marked effects on afferent but not efferent arterioles, Up(4)A may play a role in renal hemodynamics and possibly blood pressure regulation.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells.

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Dermatopathic lymphadenopathy: a differential diagnosis of enlarged lymph nodes in uremic pruritus.

BACKGROUND: In end-stage renal disease patients, the incidence of both infections and malignancies is increased leading to a higher incidence of peripheral lymphadenopathy. In the present work we describe a rare but probably underdiagnosed cause for enlarged lymph nodes in uremic patients. PATIENT: A 43-year-old male patient was admitted to our hospital with inguinal lymphadenopathy and pruritus. He turned out to be uremic due to focal segmental glomerulosclerosis (creatinine 4.5 mg/dl, MDRD creatinine clearance 12 ml/min). FINDINGS: Sonography revealed enlarged lymph nodes (up to 4 cm) with intact corticohilar border differentiation. After extirpation of an inguinal lymph node, histological examination established the diagnosis of dermatopathic lymphadenopathy. T cell lymphoma was excluded by PCR for T cell receptor-gamma rearrangements and subsequent GeneScan analysis. Intravenous fluid supplementation with subsequent decline of creatinine, UVB treatment, clemastine, and topical use of emollients led to a relief of the uremic pruritus and the lymph nodes' size normalized within 8 weeks. CONCLUSION: Dermatopathic lymphadenopathy refers to the reactive condition seen in lymph nodes that drain areas with disruption of the skin integrity, e.g. due to scratch marks. The present case report describes dermatopathic lymphadenopathy as a harmless cause of enlarged lymph nodes in uremic pruritus for the first time. This entity should be considered in the differential diagnosis of peripheral lymphadenopathy of unknown origin in patients with renal failure.

Mesangial IgA deposition in minimal change nephrotic syndrome: coincidence of different entities or variant of minimal change disease?

BACKGROUND: Mesangial deposition of IgA (MCA) is a very rare finding in minimal change disease and has previously been considered a pure coincidence. In the U.S. and Europe only anecdotal case reports exist. To date, there has been no consensus on nomenclature and categorization of this entity. We describe 2 cases of MCA with analogue histological findings but relevant differences in clinical presentation, and we discuss the clinical implications of mesangial IgA deposition in minimal change nephrotic syndrome. PATIENTS: A 47-year-old female was admitted to hospital with nephrotic syndrome, microscopic hematuria, arterial hypertension and slight impairment of renal function 3 weeks after an unspecific upper airway infection. A 42-year-old male presented with nephrotic syndrome, microscopic hematuria, normotension and normal renal function. Both of the nephrotic syndromes were steroid-responsive and steroid-dependent. FINDINGS: The clinical presentation of the male patient was consistent with the features of minimal change glomerulopathy, whereas the female patient combined signs of minimal change disease and IgA nephropathy. Light microscopy revealed mesangial IgA immune deposits and slight mesangial hypercellularity. Electron microscopic studies of MCA patients disclose diffuse effacement of glomerular foot processes. CONCLUSION: Our cases and a review of the literature indicate that the histological diagnosis of MCA may comprise different pathogenetic entities. From the clinical point of view, MCA has to be regarded as a minimal change nephrotic syndrome with symptomatic or asymptomatic mesangial IgA deposition. IgA deposition constitutes a risk factor for impairment of renal function and indicates a frequently relapsing course.

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns.

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.

[BENEFIT Kidney--significance of a nephrology screening at intervention outset and therapy success]. / BENEFIT Niere -- Bedeutung eines Nephrologie-Screenings für Interventionsbeginn und Therapieerfolg.

BACKGROUND AND OBJECTIVE: Early specialist care of patients with renal disease, including timely and planned onset of dialysis, determine the course of the disease, quality of life, hospitalization and life expectancy. A multi-centre enquiry by standardized questionnaire was undertaken to define and analyse medical care of newly dialysis-requiring patients. PATIENTS AND METHODS: Data on 551 patients in five different regions of Germany who for the first time required renal replacement treatment were prospectively collected between July 2002 and March 2003. Documentation of history, clinical findings and biochemical tests was done on consecutive patients with a standardized questionnaire, until the desired number of cases was reached. RESULTS: The mean age of the patients (55.4% males) was 64.8 years. 30.7% had diabetes mellitus, 22.3% arterial hypertension/nephrosclerosis and 16.9% glomerulonephritis/vasculitis. Early predominantly nephrological care had been undertaken in 38.7% of patients. 59.0% were cared for almost exclusively by their general practitioner until the time when dialysis was started. 229 patients (41.6%) were referred to specialist (nephrologists) only when dialysis had become necessary. The onset of dialysis was at the right time in only 50.5% of this group. Comparing the care given by nephrologists with that by general practitioners, elective (i.e. planned) dialysis was begun in 81.0% vs. 48.0% (p<0.05). Hospitalization in the two groups was 54.5% vs. 83.7% (p<0.05), the duration of hospital stay 11.4 vs. 17.4 days (p<0.05). CONCLUSION: Fewer than 40% of patients with chronic renal disease in preterminal renal failure (stage IV) were under the care of nephrologists. The lower the degree of nephrological care the more frequent was there a delay in the onset of dialysis treatment. The incidence and the duration of hospital stay was longer. Structured treatment pathways and incentives need to be formulated to reduce the incidence of wrong or substandard treatment of patients with impaired renal function.

Increased plasma phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression.

NO prevents atherogenesis and inflammation in vessel walls by inhibition of cell proliferation and cytokine-induced endothelial expression of adhesion molecules and proinflammatory cytokines. Reduced NO production due to inhibition of either eNOS or iNOS may therefore reinforce atherosclerosis. Patients with end-stage renal failure show markedly increased mortality due to atherosclerosis. In the present study we tested the hypothesis that uremic toxins are responsible for reduced iNOS expression. LPS-induced iNOS expression in mononuclear leukocytes was studied using real-time PCR. The iNOS expression was blocked by addition of plasma from patients with end-stage renal failure, whereas plasma from healthy controls had no effect. Hemofiltrate obtained from patients with end-stage renal failure was fractionated by chromatographic methods. The chromatographic procedures revealed a homogenous fraction that inhibits iNOS expression. Using gas chromatography/mass spectrometry, this inhibitor was identified as phenylacetic acid. Authentic phenylacetic acid inhibited iNOS expression in a dose-dependent manner. In healthy control subjects, plasma concentrations were below the detection level, whereas patients with end-stage renal failure had a phenylacetic acid concentration of 3.49 +/- 0.33 mmol/l (n = 41). It is concluded that accumulation of phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression. That mechanism may contribute to increased atherosclerosis and cardiovascular morbidity in patients with end-stage renal failure.

Identification of dinucleoside polyphosphates by matrix-assisted laser desorption/ionisation post-source decay mass spectrometry.

Dinucleoside polyphosphates are a group of intra- and extracellular mediators controlling numerous physiological functions. In this study dinucleoside polyphosphates were examined by positive ion matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MADLI-TOFMS). 3-Hydroxypicolinic acid was used as UV-absorbing matrix. For the individual dinucleoside polyphosphates Ap(n)A (n = 2-7), Ap(n)G (n = 2-6) and Gp(n)G (n = 2-6), MALDI post-source decay (PSD) mass spectra were measured. Each mass peak in the MALDI-PSD mass spectra could be assigned to individual fragments of dinucleoside polyphosphates. The comparison of the fragmentation patterns of the dinucleoside polyphosphates presented here demonstrates that dinucleoside polyphosphates preferably cleave to fragment ions consisting of the corresponding mononucleoside polyphosphates as well as the corresponding nucleosides and bases during flight in the field-free drift path of the MALDI mass spectrometer. Therefore, the MALDI-PSD approach described here is suitable for identification of other dinucleoside polyphosphates. The present MALDI-PSD mass spectra may be used as MALDI-PSD mass reference spectra for future identification of dinucleoside polyphosphates and other nucleotides.

Identification of dinucleoside polyphosphates in adrenal glands.

Dinucleoside polyphosphates have been characterised as extracellular mediators controlling numerous physiological functions like vascular tone or cell proliferation. Here we describe the isolation and identification of dinucleoside polyphosphates Ap(n)A (with n=2-3), Ap(n)G (with n=2-6) as well as Gp(n)G (with n=2-6) from adrenal glands. These dinucleoside polyphosphates are localised in granules of the adrenal glands. The dinucleoside polyphosphates diadenosine diphosphate (Ap(2)A), diadenosine triphosphate (Ap(3)A), adenosine guanosine polyphosphates (Ap(n)G) and diguanosine polyphosphates (Gp(n)G), both with phosphate group (p) numbers (n) ranging from 2 to 6, were identified by fractionating them to homogeneity by preparative size-exclusion- and affinity-chromatography as well as analytical anion-exchange and reversed-phase-chromatography from deproteinised adrenal glands and by analysis of the homogeneous dinucleoside polyphosphates containing fractions with post-source-decay (PSD) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). The identity of the dinucleoside polyphosphates was confirmed by retention time comparison with authentic dinucleoside polyphosphates. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5(')-positions of the riboses in all dinucleoside polyphosphates purified from adrenal glands. In conclusion, the identification of these dinucleoside polyphosphates in adrenal gland granules emphasises that these dinucleoside polyphosphates can be released from the adrenal glands upon stimulation into the circulation.

Selective agonism of group I P2X receptors by dinucleotides dependent on a single adenine moiety.

We have investigated the activity of naturally occurring high-performance liquid chromatography-purified diadenosine polyphosphates (Ap(n)A, n = 5-6), adenosine polyphospho guanosines (Ap(n)G, n = 5-6), and diguanosine polyphosphates (Gp(n)G, n = 5-6) under voltage-clamp conditions at recombinant rat P2X1-4 purinoceptor subtypes expressed in Xenopus laevis oocytes. At rP2X1 and rP2X3 receptors, Ap(n)As and Ap(n)Gs evoked concentration-dependent inward currents. Gp(n)Gs were not active at these receptors. At rP2X2 and rP2X4 receptors, dinucleotides did not show significant activity. For the rP2X1 receptor, Ap(n)As and Ap(n)Gs were partial agonists; for the P2X3 receptor, only Ap5G was full agonist, whereas the other tested substances were partial agonists. The rank order of potency at rP2X1 was ATP > or = Ap6A > or = Ap5A > or = Ap6G > or = Ap5G, and rank order of efficacy was ATP > or = Ap5A > or = Ap6A > Ap5G > Ap6G, whereas at rP2X3 the rank order of potency was ATP > Ap5G > or = Ap5A > or = Ap6A > or = Ap6G and the rank order of efficacy was ATP approximately Ap5G > or = Ap5A approximately Ap6A > or = Ap6G. For rP2X1 and rP2X3 it is evident that receptor agonism depended on the presence of at least one adenine moiety in the dinucleotide, while the presence of a guanine moiety had a significant impact and decreased agonist efficacy. The data suggest that naturally occurring Ap(n)As and Ap(n)Gs may play an important physiological role in different human tissues and systems by activating group I P2X receptors.

Homocyst(e)ine metabolism in hemodialysis patients treated with vitamins B6, B12 and folate.

Hyperhomocyst(e)inemia is commonly accepted as an independent atherosclerotic risk factor. In most hemodialysis patients, serum homocyst(e)ine is markedly elevated and may contribute to premature atherosclerosis in these patients. Whereas the beneficial effect of folate supplementation on serum homocyst(e)ine has been extensively studied, there are less detailed studies on the effects of cobalamin and pyridoxal phosphate alone, or in combination with folate. We examined the effect of a four-week course of intravenous treatment with folate (1.1 mg), cobalamin (1.0 mg), and pyridoxal phosphate (5.0 mg), administered once (group 1), twice (group 2) or thrice (group 3) weekly in 33 hemodialysis patients divided in three groups of 11 patients. All patients were followed for a further four weeks after treatment was stopped. Serum homocyst(e)ine, cobalamin, folate and pyridoxal phosphate, as well as the metabolites of homocyst(e)ine, methylmalonate, 2-methylcitrate and cystathionine, were determined before, during and after treatment. Baseline serum homocyst(e)ine correlated significantly with serum folate (P=0.0149), cobalamin (P=0.0047) and pyridoxal phosphate (P=0.0408). Correlations independent from the other metabolites or vitamins were found for methylmalonate (P=0.003) and folate (P=0.029). All regimens increased serum cobalamin significantly (in group 1 from 444 +/- 215 to 17,303 +/- 11,989 pg/ml, P<0.01; in group 2 from 542 +/- 633 to 44,896 +/- 15,772 pg/ml, P<0.001; in group 3 from 548 +/- 394 to 77,961 +/- 31,546 pg/ml, P<0.001), but did not increase any of the other vitamin levels. Serum homocyst(e)ine was lowered significantly by 39.8% +/- 31.9% (P<0.05) with two and by 30.1% +/- 26.9% (P<0.05) with three vitamin dosages weekly, but not with one dosage weekly. Since methylmalonate is known to be a sensitive marker of cobalamin deficiency, the data support an important influence of cobalamin levels on baseline homocyst(e)ine levels. Increasing cobalamin levels and additional treatment with folate and pyridoxal phosphate 156 may decrease serum homocyst(e)ine in the same way as high doses of folate alone.