The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.

Properties and regulation of organic cation transport in freshly isolated human proximal tubules.

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.

Impact of estradiol blood concentrations on skin capillary Laser Doppler flow in premenopausal women.

OBJECTIVE AND METHOD: Estradiols are known to prevent cardiovascular diseases, but the underlying mechanisms remain unclear. We assessed the finger skin capillary blood flow in 20 premenopausal women with oral contraception noninvasively by Laser Doppler flowmetry (LDF). Blood flow was registered both under basal conditions and after 2 min of arterial occlusion. Furthermore, the combination with a beat-to-beat online finger blood pressure monitor allowed the calculation of the regional peripheral resistance (RPR). All measurements were performed in the menstrual phase. LDF and RPR were correlated to the estradiol whole blood levels. RESULTS: Both baseline (r(2)=0.826; P<0.01) and vasodilation (r(2)=0.747; P<0.01) blood flux were significantly correlated to the corresponding estradiol concentrations. A multiple stepwise regression analysis (with age, height, weight, duration of oral contraception, nicotine pack years and estradiol levels in the equation) showed positive correlations between estradiol levels and (a) baseline (P<0.05) or (b) vasodilation (P<0.05) Laser Doppler flux or (c) time to reach the peak flux (r(2)=0.31; P<0.05). Furthermore, the minimal local vascular resistance was negatively correlated to the estradiol levels (r(2)=0.45; P<0.05). CONCLUSION: The study provides evidence that both the baseline and the vasodilation capillary blood flux in women in the menstrual phase depend on the estradiol whole blood levels. Furthermore, the local vascular resistance shows a negative correlation to the hormonal state. Functional or structural alterations of the microvasculature may therefore contribute to the estradiol-associated cardiovascular protection.

Diadenosine polyphosphates activate a Ca(2+)-dependent K(+)-conductance in porcine aortic smooth muscle cells via P2-purinoceptors.

Effects of the diadenosine polyphosphates P(1),P(3)-diadenosine triphosphate (Ap3A), P(1),P(4)-diadenosine tetraphosphate (Ap4A), P(1),P(5)-diadenosine pentaphosphate (Ap5A) and P(1), P(6)-diadenosine hexaphosphate (Ap6A) and of adenosine, ATP, ADP, AMP, UTP on smooth muscle cells from porcine aorta were examined. Membrane voltages and cellular conductances were measured in the slow whole cell configuration of the patch clamp technique. All four diadenosine polyphosphates, adenosine, AMP and ADP predominantly hyperpolarized membrane voltages with only occasional transient initial depolarizations whereas ATP and UTP led to sustained depolarizations. All four diadenosine polyphosphates increased cellular conductances. The effects of Ap5A on membrane voltages were almost completely inhibited by the putative P2-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 micromol/l) and only partially reduced by the putative A(2)-purinoceptor antagonist 3,7-dimethyl-1-propragyl-xanthine (DMPX, 10 micromol/l) or the Ap4A-receptor antagonist diinosine pentaphosphate (Ip5I, 10 micromol/l). The adenosine-induced hyperpolarization was partially reduced by the putative A(1)-purinoceptor antagonist 8-cyclopentyl-1,3-dipropargylxanthine (DPCPX, 0.1 micromol/l) or by DMPX while PPADS or Ip5I were without effects. Ap5A-induced hyperpolarizations were inhibited by Ba(2+) and clotrimazole but not by glibenclamide. We conclude that diadenosine polyphosphates activate predominantly a Ca(2+)-dependent K(+)-conductance in smooth muscle cells obtained from porcine aorta most likely mediated via P2Y-purinoceptors and possibly partially also by Ap4A receptors.

The beneficial effects of calcium channel blockers on long-term kidney transplant survival are independent of blood-pressure reduction.

There is a growing body of evidence suggesting that calcium channel blockers (CCB) exert beneficial effects on kidney transplant survival. However, it is not completely understood if these agents act independently of blood-pressure reduction. In the present study, the 5-yr follow-up of 45 kidney transplant recipients receiving CCB during the 60-month follow-up period was compared to that of recipients with lower blood pressure and an antihypertensive treatment without CCB. During the whole follow-up, systolic (127.4 +/- 2.5 vs. 139.4 +/- 2.1 mmHg, p < 0.05) as well as diastolic blood pressure (78.8 +/- 1.1 vs. 84.8 +/- 1.8 mmHg, p < 0.05) was higher in the group receiving CCB. Moreover, in CCB-treated recipients, a significant (p < 0.05) higher increase in proteinuria was detected (from 759 +/- 120 to 1690 +/- 359 mg/24 h vs. 180 +/- 45 to 340 +/- 45 mg/24 h). Despite higher blood pressure and higher proteinuria, the increase in serum creatinine in the group of CCB-treated recipients was significantly lower (0.01 mg/dL/month) in comparison to that of the controls (0.02 mg/dL/month, p < 0.05). Moreover, the 5-yr transplant survival was significantly higher in CCB-treated recipients (62.3 vs. 31.8%, p < 0.05). The results of the present study further support the beneficial effects of CCB in kidney transplant recipients, which are independent of blood-pressure reduction.

Effects of moxonidine and clonidine on potassium excretion in Sprague-Dawley rats.

Recently we demonstrated that clonidine and moxonidine exert specific action on fractional fluid and Na+ excretion in anaesthetised Sprague-Dawley rats. Classically, most of the diuretics used induce an increased K+ excretion, at least in part due to Na+ load in the distal tubule and exchange of Na+ by K+. Therefore, we studied the effects of moxonidine and clonidine on K+ excretion in anaesthetised Sprague-Dawley rats. Moxonidine (0.25 and 0.5 mg kg-1 body wt. i.v.) increased transiently K+ (1.0 +/- 0.3 -1.9 +/- 0.4 and 0.9 +/- 0.2 -2.9 +0.7 micromol min-1 100 g body wt.) and Na+ (1.4 +/- 1.0 -6. 9 +/- 3.1 and 0.8 +/- 0.36 -6.6 +/- 1.5 micromol min-1100 g body wt.) excretion. Clonidine (0.25 mg kg-1) caused a more pronounced increase in K+ (1.0 +/- 0.1 -2.7 +/- 0.4 micromol min-1 100 g body wt.) and Na+ (0.6 +/- 0.3 -9.5 +/- 0.4 micromol min-1 100 g body wt.) excretion, whereas the higher dose of 0.5 mg kg-1 body wt. had less effect as compared to moxonidine (K+: 0.8 +/- 0.1 -1.7 +/- 0.2 micromol min-1 100 g body wt., Na+: 0.3 +/- 0.1 -3.4 +/- 1.0 micromol min-1 100 g body wt.). The increased electrolyte excretion returned (similar to moxonidine) to baseline levels within 20 min after injection of the drugs. Antagonists such as idazoxan and yohimbine did not change K+ and Na+ excretion by their own. Both, the non-selective imidazoline/alpha2-adrenoceptor antagonist idazoxan and the pure alpha2-adrenoceptor antagonist yohimbine attenuated the moxonidin-induced effects on K+ and Na+ excretion. This could be also observed with clonidine and simultaneous injection of these two antagonists. Our results demonstrate that moxonidine and clonidine also increase renal K+ excretion in this animal model. K+and Na+ excretion show a parallel behaviour, indicating that the increased K+ excretion is mainly due to Na+ load in the tubular system.

Hypochromic red cells and reticulocyte haemglobin content as markers of iron-deficient erythropoiesis in patients undergoing chronic haemodialysis.

BACKGROUND: In patients on chronic haemodialysis, because of a non-specific increase in serum ferritin, iron deficiency may be overlooked leading to failure of erythropoietin treatment. A reticulocyte haemglobin content < 26 pg and a percentage of hypochromic red cells > 2.5 have been proposed as markers of iron-deficient erythropoiesis in such subjects, but it is unclear which parameter is superior. METHODS: We measured haematocrit, reticulocyte haemglobin content, ferritin and the percentage of hypochromic red cells over 10-150 days in 36 chronic haemodialysis patients in a university hospital. Transferrin saturation was also measured in a subset of 25 patients; iron deficiency was defined as a transferrin saturation < 15%. RESULTS: The diagnostic sensitivity and specificity of a reticulocyte haemoglobin content < 26 pg in detecting iron deficiency were 100% and 73% respectively, compared with 91% and 54% for a percentage of hypochromic red cells > 2.5. Paradoxical reticulocyte haemglobin concentrations occurred on follow-up in five patients receiving 4000 U erythropoietin per haemodialysis (HD). In three patients, reticulocyte haemglobin content exceeded 26 pg despite a persistent lack of iron. In a fourth, iron gluconate (62.5 mg i.v./HD) increased transferrin saturation to 27% and reduced the percentage of hypochromic red cells from 12 to 4, while reticulocyte haemglobin remained > 30 pg. In the final patient, iron gluconate increased transferrin saturation from 8 to 30% and reduced the percentage of hypochromic red cells from 40 to below 5, but reticulocyte haemglobin content remained < or = 26 pg throughout. CONCLUSIONS: The reticulocyte haemglobin content is superior to the percentage of hypochromic red cells in detecting iron deficiency in haemodialysis patients.

Serum antibody titers in a systemic lytic therapy with streptokinase.

BACKGROUND: Anaphylactic reactions to streptokinase are rare but potentially life-threatening complications. Gamma E immunoglobulin (IgE) mediated mechanisms, probably due to streptococcal infections, have been implicated. We investigated the value of in vitro laboratory or dermatologic tests in predicting anaphylactic reactions due to streptokinase and the value of antistreptolysin titers (ASL) in predicting the amount of specific IgE (sIgE) and specific gamma G immunoglobulin (sIgG) neutralizing antibodies to streptokinase. METHODS: We measured serum levels of total IgE, streptokinase sIgE and sIgG, and ASL in 16 patients before and 9 and 41 days after streptokinase therapy. Immediately before therapy, intracutaneous testing with 100 IU streptokinase was done. RESULTS: Dermatologic testing did not identify patients prone to allergic reactions. Moreover, not all patients with increased sIgE levels had allergic reactions. These reactions were independent of the dose of streptokinase given. In spite of steroid prophylaxis, allergic reactions occurred in 3 of 16 patients, but none showed life-threatening anaphylaxis. Streptokinase sIgE and sIgG concentrations were closely related to ASL titers. CONCLUSIONS: Plasma levels of sIgG, sIgE, and ASL titers showed a good correlation. We believe ASL titers can be used for the estimation of neutralizing antibodies instead of streptokinase sIgG antibodies. Currently, no laboratory or dermatologic test allows reliable predictions of allergic reactions to streptokinase.

Effects of extracellular cadmium on renal basolateral organic anion transport.

Heavy metal ions are well-known nephrotoxic agents. Usually, they induce a damage of various renal transport systems. Cd2+, however, has been shown to enhance renal basolateral organic cation transport. Therefore, the effects of Cd2+ on the basolateral p-aminohippuric acid (PAH) uptake of microdissected nonperfused rabbit kidney S2 proximal tubule segments were investigated. Incubation with Cd2+ induced a bell-shaped concentration response curve with a 2-fold increase of the PAH transport at a Cd2+ concentration of 10(-6) M. Since Cd2+ has been described to activate protein kinase C (PKC), the effects of the PKC inhibitor staurosporine were also tested. Staurosporine (10(-8) M) could, however, not reduce the effects of Cd2+. Cd2+ may exert its effects by an as yet unknown mechanism that is different from the PKC-activating mechanism of phorbol esters. A stimulation of the tricarboxylic acid cycle in kidney cells by Cd2+ may, however, increase the delivery of alpha-ketoglutarate. Indeed, incubation of proximal tubules with alpha-ketoglutarate increased PAH transport across the basolateral membrane significantly. Thus, the observed effects of Cd2+ may be due to an enhanced transport of p-aminohippuric acid by stimulation of exchange of PAH with alpha-ketoglutarate. The modulation of renal organic anion transport may be another aspect of Cd2+ nephrotoxicity.

The surface roughness of restorative materials and dental tissues after polishing with prophylaxis and polishing pastes.

The effect of prophylaxis and polishing pastes on bovine tooth hard tissues and dental materials was investigated in vitro. Four restorative dental materials (gold, amalgam, a conventional composite, a microfilled composite), brought to a standard surface finish, and two bovine dental hard tissues (enamel, dentin) were polished with ten different prophylaxis and polishing pastes. Before polishing, SEM photographs were taken of all materials, and surface profiles were obtained of every sample. After polishing, surface profiles from every sample and SEM photographs of every material/paste combination were obtained. Using the same techniques, two-step polishing procedures were also investigated. The evaluation of the surface profiles and the rated SEM photographs (by 20 dentists) showed significant paste and material differences. There was a significant correlation (0.82) between the results of the surface profiles and the SEM ratings. Almost all pastes roughened the surfaces of dental restorative materials and only a few pastes showed a tendency to render the surfaces of dental restorative materials and only a few paste showed a tendency to render the surfaces of dental hard tissues smoother. Therefore, if polishing is indicated after periodontal treatment, the paste used should be selected carefully. Since the surface roughness of conventional composites increases tremendously during each polishing with pastes, their surfaces should be avoided when using polishing pastes. If polishing with a paste in indicated, conventional composites should be polished afterwards with aluminium silicate coated discs.