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E Schlatter

Publications and source records attributed to E Schlatter.

At least 19 recordsLinked to original sources
PubMed2002

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.

Animals↗
PubMed2001

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat.

BACKGROUND: Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS: RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS: GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION: GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

Animals↗
PubMed2001

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.

Biological Transport↗
PubMed2001

Cellular localization of the potassium channel Kir7.1 in guinea pig and human kidney.

BACKGROUND: K(+) channels have important functions in the kidney, such as maintenance of the membrane potential, volume regulation, recirculation, and secretion of potassium ions. The aim of this study was to obtain more information on the localization and possible functional role of the inwardly rectifying K(+) channel, Kir7.1. METHODS: Kir7.1 cDNA (1114 bp) was isolated from guinea pig kidney (gpKir7.1), and its tissue distribution was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, a genomic DNA fragment (6153 bp) was isolated from a genomic library. cRNA was expressed in Xenopus laevis oocytes for functional studies. Immunohistochemistry and RT-PCR were used to localize Kir7.1 in guinea pig and human kidney. RESULTS: The expression of gpKir7.1 in Xenopus laevis oocytes revealed inwardly rectifying K(+) currents. The reversal potential was strongly dependent on the extracellular K(+) concentration, shifting from -14 mV at 96 mmol/L K(+) to -90 mV at 1 mmol/L K(+). gpKir7.1 showed a low affinity for Ba(2+). Significant expression of gpKir7.1 was found in brain, kidney, and lung, but not in heart, skeletal muscle, liver, or spleen. Immunocytochemical detection in guinea pig identified the gpKir7.1 protein in the basolateral membrane of epithelial cells of the proximal tubule. RT-PCR analysis identified strong gpKir7.1 expression in the proximal tubule and weak expression in glomeruli and thick ascending limb. In isolated human tubule fragments, RT-PCR showed expression in proximal tubule and thick ascending limb. CONCLUSION: Our results suggest that Kir7.1 may contribute to basolateral K(+) recycling in the proximal tubule and in the thick ascending limb.

Amino Acid Sequence↗
PubMed2001

A novel assay for determination of diadenosine polyphosphates in human platelets: studies in normotensive subjects and in patients with essential hypertension.

OBJECTIVE: Diadenosine polyphosphates (APnAs, n = 3-6) are a family of endogenous vasoactive purine dinucleotides which have been isolated from thrombocytes. Diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) are more potent than diadenosine tetraphosphate (AP4A) and diadenosine triphosphate (AP3A) and cause skeletal muscle vasoconstriction in rats. Little is known about their physiological and pathophysiological significance in humans. The aims of the present study were to compare thrombocyte APnA concentrations in patients with essential hypertension (HYP) and in healthy normotensive humans (CON) using a novel quantitative assay and to assess a possible relationship between thrombocyte APnA concentrations and skeletal muscle vascular resistance. DESIGN AND METHODS: We describe a novel assay for quantification of APnAs in human platelets, involving platelet isolation from human blood, a solid-phase extracting procedure with a derivatized resin, desalting and quantitative determination of the substances with an ion-pair reversed-phase high-performance liquid chromatography (HPLC) system. The structural integrity of the isolated APnAs was confirmed by mixed assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) measurements and co-elution with added standards. The detection threshold for all four APnAs was 1 pmol/l and the inter-assay coefficients of variation were < 11% (n = 12). After venous blood sampling, mean arterial blood pressure (MAP) and forearm blood flow (FBF, using venous occlusion plethysmography) were measured in HYP and CON. Forearm vascular resistance (FVR) was calculated as MAP/FBF. significantly differ in platelet AP3A and AP4A content, but HYP had significantly higher thrombocyte concentrations of AP5A (56 +/- 7 versus 32 +/- 3 ng/microg beta-thromboglobulin, P = 0.003) and AP6A (10 +/- 1 versus 6 +/- 1 ng/microg beta-thromboglobulin, P = 0.015) than CON. HYP had significantly elevated FVR (50 +/- 6 versus 33 +/- 2 arbitrary units, P = 0.01) compared to CON. Significant correlations were found between AP5A and FVR (p = 0.38, P = 0.04) as well as between AP6A and FVR (p = 0.42, P = 0.02). In contrast, there were no significant correlations between APnAs and MAP. CONCLUSIONS: The study shows that thrombocyte concentrations of AP5A and AP6A are elevated in patients with essential hypertension. Vasoconstriction caused by release of AP5A and AP6A from thrombocytes may contribute to the increase of vascular resistance in hypertensive patients.

Adult↗
PubMed2001

cGMP serves as an extracellular regulator of a Ca(2+)-dependent K(+) channel in immortalized human proximal tubule cells.

Recently we showed that a K(+) channel in immortalized human kidney epithelial (IHKE-1) cells derived from the proximal tubule is regulated by natriuretic peptides in cell-attached patches and directly regulated by cGMP in excised inside-out oriented membrane patches [1]. The patch clamp technique was used to investigate the regulatory effect of extracellular, non-membrane permeable cGMP on membrane voltage and the regulation of this K(+) channel in outside-out oriented membrane patches. In 7 out of 7 experiments the membrane voltage of IHKE-1 cells depolarized by 3.9 +/- 0.1 mV when the non-membrane permeable cGMP was added to the bath solution. In outside-out oriented membrane patches cGMP inhibited P(o) already at 1 microM (-12 +/- 4%, n=7), at 0.1 mM inhibition of P(o) reached -39 +/- 6% (n=14). cAMP (0.1 mM) only had a weak inhibitory effect (n=7). GTP and ATP (n=7 each) had no significant effect on P(o) from the outside. When cGMP was added to the pipette solution in experiments with outside-out oriented membranes cGMP still inhibited this K(+) channel from the outside by 36 +/- 6% (n=6). In 4 paired experiments 8-Br-cGMP (0.1 mM) showed a significantly higher inhibitory effect on P(o) compared to cGMP (0.1 mM). cGMP inhibits a K(+) channel in human proximal tubule cells from the outside and may serve as an autocrine and paracrine regulatory factor in the kidney.

Calcium↗
PubMed2001

High salt intake increases uroguanylin expression in mouse kidney.

The intestinal peptides, guanylin and uroguanylin, may have an important role in the endocrine control of renal function. Both peptides and their receptor, guanylyl cyclase C (GC-C), are also expressed within the kidney, suggesting that they may act locally in an autocrine/paracrine fashion. However, their physiological regulation within the kidney has not been studied. To begin to address this issue, we evaluated the distribution of uroguanylin and guanylin messenger RNA (mRNA) in the mouse nephron and the regulation of renal expression by changes in dietary salt/water intake. Expression was determined in 1) wild-type mice, 2) two strains of receptor-guanylyl cyclase-deficient mice (ANP-receptor-deficient, GC-A-/-, and GC-C-deficient mice); and 3) cultured renal epithelial (M-1) cells, by RT-PCR, Northern blotting and immunocytochemistry. Renal uroguanylin messenger RNA expression was higher than guanylin and had a different distribution pattern, with highest levels in the proximal tubules, whereas guanylin was mainly expressed in the collecting ducts. Uroguanylin expression was significantly lower in GC-C-/- mice than in GC-A-/- and wild-types, suggesting that absence of a receptor was able to down-regulate ligand expression. Salt-loading (1% NaCl in drinking water) increased uroguanylin-mRNA expression by >1.8-fold but had no effect on guanylin expression. Uroguanylin but not guanylin transcripts were detected in M-1 cells and increased in response to hypertonic media (+NaCl or mannitol). Our results indicate that high-salt intake increases uroguanylin but not guanylin expression in the mouse kidney. The synthesis of these peptides by tubular epithelium may contribute to the local control of renal function and its adaptation to dietary salt.

Animals↗
PubMed2000

Amino acid transport in the renal proximal tubule.

In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na+, Cl- and K+) or use an H+-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects.

Amino Acids↗
PubMed2000

In vivo effects of diadenosine polyphosphates on rat renal microcirculation.

BACKGROUND: Diadenosine polyphosphates (APXA) are vasoactive nucleotides that elicit effects via purinoceptors. Recent data suggest differential effects of APXA on kidney vasculature. METHODS: The in vivo effects of AP3A, AP5A, and adenosine on renal microvessels and the role of purinoceptors were investigated by the application of agonists to the hydronephrotic rat kidney and preincubation with respective antagonists. RESULTS: The addition of the agonists (10-7 mol/L up to 10-4 mol/L) resulted in a concentration-dependent transient vasoconstriction [interlobular artery (ILOB): adenosine 30 +/- 7%, N = 7, AP3A 35 +/- 10%, N = 5; AP5A 66 +/- 19%, N = 5; 10-5 mol/L each] lasting up to one minute, followed by a concentration-dependent vasodilation (ILOB: adenosine 10 +/- 3%, N = 6; AP3A 19 +/- 4%, N = 5; AP5A 12 +/- 5%, N = 6; 10-5 mol/L each). In ILOB and in the afferent arteriole (AFF), the constrictory effects of AP5A were more pronounced than those of AP3A and adenosine. In the efferent arteriole (EFF), vascular tone was only slightly affected by all agonists. The dilatory potency was comparable for all agonists in ILOB and EFF. No significant vasodilation occurred in AFF. The application of the selective A1 receptor antagonist DPCPX (10-5 mol/L) completely abolished the adenosine-induced vasoconstriction, whereas the A2 receptor antagonist DMPX and the P2 purinoceptor antagonists PPADS and A3P5P (all 10-5 mol/L) did not affect adenosine-induced constriction. The AP3A-induced constriction was abolished by DPCPX and was partially inhibited by PPADS. The constriction induced by AP5A was less sensitive to DPCPX but more sensitive to PPADS. In ILOB and EFF, DMPX or A3P5P abolished dilation after the addition of the agonists. The dilation after AP5A was not significantly reduced. In AFF, no significant dilation was observed with these agonists alone, but it was clearly visible in the presence of DPCPX or PPADS. CONCLUSIONS: APXA evoke transient constrictions in vessels of the hydronephrotic rat kidney, which are mediated by A1 and P2 purinoceptors. The length of the phosphate chain determines the degree of vasoconstriction and the extent to which the substances exert effects on the P2 purinoceptor subtypes. ILOB and AFF are more potently affected by APXA than EFF. Afferent vasodilation is partially overridden by sustained vasoconstriction.

Adenosine↗
PubMed2000

Regulation of mesangial cell apoptosis and proliferation by intracellular Ca(2+) signals.

BACKGROUND: In inflammatory glomerular diseases, proliferation, as well as apoptosis of mesangial cells (MCs), has been shown histomorphologically. Both processes may regulate the cellular content of the mesangium by closely influencing each other. In the present study, we examined whether the cytoplasmic free Ca(2+) concentration [Ca(2+)](i) is involved as a key second messenger in the regulation of proliferative and apoptotic events. METHODS: Thapsigargin, an inhibitor of the endoplasmic Ca(2+)-Mg(2+)-ATPase, was used as a test substance to investigate the role of [Ca(2+)](i) in signaling MC apoptosis and growth in vitro. Apoptosis was determined by nuclear chromatin staining with Hoechst 33258, by a [3H]-thymidine-based DNA fragmentation assay or by flow cytometry detecting binding of FITC-conjugated annexin V. Proliferation was measured by [3H]-thymidine incorporation into acid-precipitable material and corroborated by cell counting. RESULTS: Thapsigargin significantly induced apoptosis and inhibited proliferation dose dependently in nanomolar concentrations without evoking necrotic damage when administered not longer than 12 hours. Significant apoptosis was measurable after a six-hour treatment of MCs with thapsigargin. Determination of [Ca(2+)](i) by fura-2-dependent spectrofluorometry showed that thapsigargin was able to induce prolonged [Ca(2+)](i) rises that could be prevented by preincubation with the intracellular Ca(2+) chelator 1, 2-bis(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid (BAPTA) acetomethyl ester (AM). BAPTA had no influence on MC viability but reversed thapsigargin-induced apoptosis to control levels. After thapsigargin treatment (100 nmol/L, 12 hours), apoptotic MCs had a significantly higher [Ca(2+)](i) of 251 +/- 25 nmol/L (N = 41) as compared with MCs that were not or not yet apoptotic ([Ca(2+)](i) of 116 +/- 20 nmol/L, N = 26, P < 0,05). Platelet-derived growth factor (PDGF), a well-characterized growth factor for MCs, reversed the effects of thapsigargin on proliferation and apoptosis in a similar fashion as BAPTA. PDGF acutely stimulated increases of [Ca2+]i but abolished thapsigargin-dependent, but not angiotensin II- or ATP-induced Ca(2+) rises when administered during a 12-hour preincubation. CONCLUSIONS: Our data suggest that a sustained increase of [Ca(2+)](i) may serve as a signal to trigger MC apoptosis. Growth factors such as PDGF can abolish apoptosis induced by elevations of [Ca(2+)](i) by altering intracellular Ca(2+) signaling.

Animals↗
PubMed2000

Diadenosine polyphosphates and atrial natriuretic peptide are antiproliferative in rat mesangial cells.

Modulation of cell proliferation by vasoactive hormones and growth factors involves also changes in the activity of pH-regulatory transporters. In a preceeding paper (1) we examined the influence of such factors on cellular pH Here the influence of the same factors, diadenosine polyphosphates (ApnA), atrial natriuretic peptide, the growth factor PDGF and the Ca2+-ATPase inhibitor thapsigargin, on proliferation of cultured rat mesangial cells was examined by quantification of [3H]-thymidine incorporation. Mesangial cells were synchronised and growth reduced (0.5% FCS for 24 h) before experiments were started, Incubation with Ap3A, Ap4A, Ap5A or Ap6A (all 10 microM) for 24 h all reduced cell proliferation by 30 to 45%. At 0.1 and 1 microM the effects of Ap4A, Ap5A and Ap6A did not reach significance The antimitogenic effect of Ap5A was not significantly different when cells were incubated for 24, 48 or 72 h. In addition there was no significant difference between the antiproliferative effect of Ap5A in cells of the second, sixth or thirteenth passage. The growth factor PDGF-BB (0.25 nM) resulted man approximately 3-fold increase in [3H]-thymidine incorporation. This increase in proliferation could be significantly reduced by coincubation with 10 microM Ap5A. The mitogenic effect of PDGF was completely abolished in the presence of the Ca2-ATPase inhibitor thapsigargin (1 nM), which also significantly reduced basal cell proliferation by approximately 40%. Incubation of mesangial cells with 10 nM ANP for 24 h reduced basal [3H]-thymidine incorporation slightly by approximately 20% and decreased the PDGF-induced stimulation. The antimitogenic effects of these agonists is especially pronounced when cells are stimulated.

Adenosine↗
PubMed2000

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.

Animals↗
PubMed2000

Influence of purinoceptor antagonism on diadenosine pentaphosphate-induced hypotension in anesthetized rats.

Diadenosine polyphosphates (ApnA; n = 3-6) are potent vasoactive agents in isolated vessels. Information on effects of ApnA in vivo is still limited despite the fact that these compounds are starting to be used in humans. This study was designed to compare the effects of ApnA and their possible metabolites on blood pressure in vivo and to functionally identify purinoceptors involved in their action. All four ApnA and their degradation products induced a sustained drop of mean arterial blood pressure during i.v. infusion, which was fully reversible. The rank order of potency was Ap4A > or = Ap6A > Ap5A = Ap3A = ATP = ADP > AMP > or = adenosine, suggesting that the hypotensive effect is predominantly evoked by the original dinucleotides and not by their degradation products. The hypotensive effect of Ap5A was reduced by the P2X and P2Y(1) purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, the A(1) purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, and the A(2) purinoceptor antagonist 3, 7-dimethyl-1-propargylxanthine. The hypertensive effect by the prototype P2X receptor agonist alphabeta-methylene ATP was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, too. Purinoceptor antagonists reduced the maximal effects of the agonists indicating a noncompetitive inhibition. In summary, the reported vasocontractile effect of ApnA seems to be limited to isolated preparations under resting tone conditions; however, the systemic cardiovascular effects of all four ApnA are hypotensive, also making them candidates for blood pressure reduction in humans. These effects are fast in onset and easily reversible. Activation of different purinoceptors in the vasculature (most probably P2Y(1) and A(2) receptors) contributes to the Ap5A-induced decrease of mean arterial blood pressure.

Animals↗
PubMed2000

Diadenosine polyphosphates cause contraction and relaxation in isolated rat resistance arteries.

The effects of diadenosine polyphosphates (APnA; n = 3-6) and adenine nucleotides on contractile reactivity of isolated rat mesenteric resistance arteries (MrA) and superior epigastric arteries (SEA), which display a dense and sparse autonomic innervation, respectively, were evaluated. All agonists examined, except adenosine and AMP, induced contractions. The rank order of potency was similar in both arteries: alpha,beta-methylene ATP (alpha,beta-meATP) > AP5A > AP6A > AP4A > ATP > ADP > AP3A. Contractions were stable during several minutes in SEA but highly transient in MrA. They were reduced after exposure to 10 microM alpha,beta-meATP and by 10 microM of the P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid. During phenylephrine (10 microM)-induced contractions, the agonists induced a further contraction in SEA. In MrA, however, further contraction was followed by marked relaxation. The rank order of relaxing potency was comparable to that of the contractile potency of agonists. Also, the relaxing effects of APnA were blunted by 10 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid and after exposure to alpha,beta-meATP. In vitro and in vivo sympathectomy with 6-hydroxydopamine and removal of the endothelium did not modify the effects of APnA in MrA. Thus, the contractile effects of APnA in resistance arteries 1) are due to a P2X purinoceptor-mediated stimulation of the smooth muscle; 2) depend on the length of the phosphate chain; and 3) are followed by endothelium-independent relaxing effects in MrA but not SEA, which may involve receptors that are similar to those mediating contraction. The regional heterogeneity of APnA effects cannot be attributed to a direct neurogenic influence.

Adenine Nucleotides↗
PubMed2000

Heterogenous vascular effects of AP5A in different rat resistance arteries are due to heterogenous distribution of P2X and P2Y(1) purinoceptors.

In the accompanying article, we showed that AP5A displayed heterogenous vasoactive effects in rat resistance arteries. It induced a stable vasoconstriction in the superior epigastric artery (SEA) and a transient vasoconstriction in the mesenteric resistance artery (MrA). In the phenylephrine-precontracted MrA AP5A induced a marked vasorelaxation. In this study the noncompetitive inhibition of the AP5A-induced vasoconstriction with pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was found to be significantly stronger in MrA than in SEA. The nonselective P2 purinoceptor antagonist suramin inhibited AP5A-induced vasoconstriction in MrA only. The vasoconstriction by the P2X purinoceptor agonist alpha,beta-methylene ATP was inhibited by with pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid and suramin similarly to that induced by AP5A. Thus, the AP5A-induced vasoconstriction is due to P2X receptor activation, but two different P2X receptors seem to be operational in the two different vessels. The AP5A-induced vasorelaxation of phenylephrine-precontracted MrA was inhibited by the P2Y(1) receptor antagonist ADP3'5'. The vasorelaxation induced by ADPbetaS (P2Y(1) agonist) also was inhibited by ADP3'5'. These findings suggest that AP5A-induced vasorelaxation of MrA is caused by P2Y(1) receptor activation. The P1 (A(2)) receptor antagonist 3, 7-dimethyl-1-propargylxanthine only slightly inhibited AP5A-induced vasorelaxation at high concentrations. Adenosine and the A(2) receptor agonist CGS21680 failed to produce significant vasorelaxation. Therefore, vasorelaxation in MrA does not involve A(2) purinoceptor activation. AP5A-induced vasorelaxation was not inhibited by Ca(2+)- or ATP-dependent K(+) channel blockade with clotrimazole, apamin, or glibenclamide. These data indicate that vasoconstriction in MrA and SEA by AP5A is due to different P2X receptors, and vasorelaxation in precontracted MrA is due to P2Y(1) receptor activation.

Animals↗
PubMed1999

A novel cGMP-regulated K+ channel in immortalized human kidney epitheliall cells (IHKE-1).

1. K+ channels from the apical membrane of immortalized human kidney epithelial (IHKE-1) cells were investigated in the cell-attached membrane configuration as well as in excised membranes using the patch clamp technique. 2. In cell-attached membrane patches the open probability (Po) of the K+ channel was 0.42 +/- 0.06 (mean +/- s.e.m. , n = 22) and its conductance was 94 +/- 5 pS with 145 mM K+ in the pipette (n = 25). In excised membrane patches the Po of the channel was 0.55 +/- 0.03 (n = 86) and its conductance was 65 +/- 2 pS (n = 68) with 145 mM K+ on one side of the membrane and 3.6 mM K+ on the other. The I-V curve of the K+ channel was not rectifying. 3. The channel was inhibited by several blockers of K+ channels such as 1 mM Ba2+ (cell-attached membrane: 78 +/- 8 %, n = 9; excised: 80 +/- 4 %, n = 26), 10 mM TEA+ (excised inside-out: 48 +/- 5 %, n = 34; excised outside-out: 100 +/- 0 %, n = 26), 0.1 mM verapamil (excised: 73 +/- 9 %, n = 12), and 10 nM charybdotoxin (excised outside-out: 67 +/- 9 %, n = 9). 4. The K+ channel was activated by depolarization and rising cytosolic Ca2+. Half-maximal activity occurred at a cytosolic Ca2+ concentration of 200 nM. In the cell-attached membrane configuration the K+ channel was inhibited in a concentration-dependent manner by atrial natriuretic peptide (ANP). Powas blocked equally well by 10 nM ANP (52 +/- 7 %, n = 10), brain natriuretic peptide (BNP; 37 +/- 11 %, n = 6) and C-type natriuretic peptide (CNP; 44 +/- 13 %, n = 8). 8-Bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.1 mM) also inhibited Poof this K+ channel, by 70 +/- 10 % (n = 5). 5. In excised membrane patches cGMP inhibited Po of this K+ channel in a concentration-dependent manner. The first significant effects were measured at a concentration of 1 microM (22 +/- 7 %, n = 6), and greatest effects were obtained at 0.1 mM (34 +/- 5 %, n = 15). cAMP (0.1 mM, n = 5) as well as GTP (0.1 mM, n = 5) had no significant effects on Po of this K+ channel. ATP (0.1 mM) had a weak inhibitory effect (17 +/- 5 %, n = 14). Addition of Mg-ATP to cGMP did not increase the inhibitory effect (30 +/- 4 %, n = 14). KT5823 (1 microM), a specific inhibitor of cGMP-dependent protein kinases, did not significantly alter the cGMP-induced reduction in Po of the K+ channel in three excised membrane patches. 6. The results present the first electrophysiological characterization of a mammalian K+ channel that is directly regulated by cGMP.

Atrial Natriuretic Factor↗
PubMed1999

Natriuretic peptides and diadenosine polyphosphates modulate pH regulation of rat mesangial cells.

Modulation of cell proliferation has often been thought to be connected to changes in the activity of pH-regulatory transporters and consequently intracellular pH (pH(i)). The influence of natriuretic peptides, diadenosine polyphosphates, adenosine and ATP as well as platelet-derived growth factor (PDGF) on pH(i) regulation of cultured rat mesangial cells was examined with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The inhibitors of Na(+)/H(+) exchange, amiloride and HOE694, blocked pH(i) recovery completely in the absence of and by approximately 50% in the presence of HCO(3)(-)/CO(2). Natriuretic peptides (ANP, BNP, CNP, urodilatin) completely inhibited pH(i) recovery in the absence of and by approximately 40% in the presence of HCO(3)(-)/CO(2). These effects were abolished by the cGMP-dependent protein kinase inhibitor KT5823. Diadenosine polyphosphates (Ap3A-Ap6A), ATP and adenosine also inhibited pH(i) recovery completely in the absence of and partially (30-40%) in the presence of HCO(3)(-)/ CO(2). The effect of adenosine was abolished in the presence of the cAMP-dependent protein kinase inhibitor KT5720, and that of Ap5A by the protein kinase C inhibitor calphostin C. PDGF activated acid extrusion in these cells by approximately 40%. From the four cloned isoforms of the Na(+)/H(+) exchanger in the rat, only transcripts of NHE-1 were found in these mesangial cell cultures using RT-PCR analysis. These data suggest that in these rat mesangial cells the Na(+)/H(+) exchanger, specifically the NHE-1 isoform, accounts for around 50% of pH(i) recovery from an acid load under physiological conditions, and that Na(+)/H(+) exchange stimulated by acidification can be inhibited by activation of PKG, PKA, and PKC and stimulated by PDGF after acute exposition to these agonists.

Adenine Nucleotides↗