Cyclic GMP stimulation by vasopressin in LLC-PK1 kidney epithelial cells is l-arginine-dependent

Summary The l-arginine antagonist N^G-monomethyl-l-arginine has been shown to inhibit nitric oxide formation from l-arginine in endothelial cells. In the present study N^G-monomethyl-l-arginine was used to assess the role of l-arginine for cyclic GMP stimulation by vasopressin in a kidney epithelial cell line (LLC-PK₁). Preincubation of cells with 1 mol/l, 10 mol/l and 100 mol/l N^G-monomethyl-l-arginine decreased cyclic GMP stimulation at 1 mol/l vasopressin by 25%, 71% and 90%, respectively. This inhibition by N^G-monomethyl-l-arginine was markedly reduced by l-arginine (2 mmol/1) but not d-arginine (2 mmol/1). Cyclic GMP stimulation by the calcium ionophore A23187 was also inhibited by N^G-monomethyl-l-arginine and enantioselectively restored by l-arginine. However, N^G-monomethyl-l-arginine did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that, in kidney epithelial cells, vasopressin induces nitric oxide formation from l-arginine leading to activation of soluble guanylate cyclase. It is concluded that nitric oxide formation from l-arginine is not only responsible for endothelium-dependent relaxation but may be a more general pathway with regulatory function for intracellular guanylate cyclase activity.Send offprint requests to K. Schror at the above address     

Clinical potential of nitric oxide-independent soluble guanylate cyclase activators

A major problem with using nitrates in the treatment of ischemic heart disease is that tolerance develops to their vasodilatory actions. YC-1 was used as the lead compound to synthesize further nitric oxide-independent soluble guanylate cyclase activators, including BAY-41-2272 and BAY-41-8543. A nitric oxide and heme-independent activator of soluble guanylate cyclase, BAY-58-2667, was subsequently discovered by high-throughput screening. Tolerance to the vasodilatory actions of BAY-41-8543 and BAY-58-2667 does not develop. Results from animal studies have suggested that these compounds may have potential in the treatment of ischemic heart disease, essential and pulmonary hypertension, congestive heart failure, glomerulonephritis and erectile dysfunction.     

Cisplatin-induced toxicity in LLC-PK1 kidney epithelial cells: role of basolateral membrane transport.

Cytotoxicity of cisplatin was evaluated after apical and/or basolateral treatment of LLC-PK1 cell monolayers grown on porous membrane filters with 300 microM cisplatin. When LLC-PK1 cells were exposed from basolateral side for 0.5-4 h, lactate dehydrogenase (LDH) release into culture medium was markedly stimulated. However, apical treatment of the cells with cisplatin for 0.5 h did not stimulate LDH release. gamma-Glutamyltransferase activity and amount of protein in the cell homogenate were markedly decreased by basolateral treatment with cisplatin. However, in the apical treatment with cisplatin, these changes were relatively small, suggesting that degrees of the toxicities were different between basolateral and apical treatment with cisplatin. Cellular platinum level after basolateral treatment with cisplatin was higher compared to that following apical treatment. Furthermore, both accumulation and toxicity of cisplatin in LLC-PK1 cells were decreased by treatment with cisplatin at 4 degrees C. These results suggested that there were specific mechanisms mediating cisplatin uptake at the basolateral membranes of LLC-PK1 cells.     

Quercetin,a phytoestrogen and dietary flavonoid,activates different membrane-bound guanylate cyclase isoforms in LLC-PK1 and PC12 cells

Accumulated evidence suggests that quercetin, a dietary flavonoid, has beneficial effects in protection against cardiovascular diseases and in the inhibition of tumour growth. We have recently shown that antioxidants such as 17beta-estradiol, resveratrol, dithiothreitol and vitamin C activate membrane-bound guanylate cyclase GC-A, a receptor for atrial natriuretic factor (ANF). Since quercetin is a phytoestrogen and potent antioxidant, it is possible that it may activate GC-A or other guanylate cyclase isoforms. We examined whether quercetin activates GC-A or GC-B (the receptor for C-type natriuretic peptide, CNP) in PC12 and porcine kidney proximal tubular LLC-PK1 cells. The results showed that quercetin activated a guanylate cyclase isoform in both cell types. Quercetin inhibited CNP-stimulated GC-B activity, but had little effect on ANF-stimulated GC-A activity in PC12 cells, suggesting that quercetin mainly activates GC-B in PC12 cells. In contrast, CNP had no effect on guanylate cyclase activity in LLC-PK1 cells, indicating that GC-B is not expressed in LLC-PK1 cells. Furthermore, quercetin had a small effect on ANF-stimulated GC-A activity and had no effect on soluble guanylate cyclase activity in LLC-PK1 cells, suggesting that quercetin does not activate GC-A, GC-B or soluble guanylate cyclase in LLC-PK1 cells. However, quercetin did stimulate membrane-bound guanylate cyclase activity in LLC-PK1 cell membranes. These results indicate that quercetin activates the GC-B isoform in PC12 cells, but activates an unknown membrane-bound guanylate cyclase isoform in LLC-PK1 cells.     

Inhibitors of cytochrome P-450 reduce cyclic GMP stimulation by glyceryl trinitrate in LLC-PK1 kidney epithelial cells

Summary The inhibitor of cytochrome P-450 cimetidine was used to asses the role of cytochrome P-450-dependent enzymes for cyclic GMP stimulation by glyceryl trinitrate in a kidney epithelial cell line (LLC-PK₁). Pretreatment of the cells with 0.1 mmol/1 cimetidine markedly decreased cyclic GMP stimulation by glyceryl trinitrate (0.03 –1 mol/l). In the presence of 0.1 mmol/1 cimetidine, the 14-fold cyclic GMP stimulation observed at 1 mol/l glyceryl trinitrate was reduced by 66%. Glyceryl trinitrate-induced cyclic GMP stimulation remained unaltered by ranitidine (0.1 mmol/1), which has a much lower affinity for the cytochrome P-450 enzyme system. Another inhibitor of cytochrome P-450, miconazole (0.1 mmol/1), also attenuated glyceryl trinitrate-induced cyclic GMP stimulation. In contrast, cimetidine and miconazole did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that in intact cells, glyceryl trinitrate-induced cyclic GMP stimulation is dependent on cytochrome P-450 enzymes which may be relevant for nitric oxide formation from organic nitrates.Send offprint requests to K. Schrör at the above address     

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).     

Chloride channel blockers decrease intracellular pH in cultured renal epithelial LLC-PK1 cells.

The effects of chloride channel blockers upon intracellular pH (pHi) were examined in renal epithelial monolayers of LLC-PK1 cells. A significant intracellular acidification was found with addition of 100 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), niflumic acid, flufenamate and diphenylamine-2-carboxylate (DPC) but not with 4,4'-diisothiocyanatostilbene-2-2'disulphonic acid (DIDS). The effects of these agents upon pHi was dose-dependent with apparent K0.5 values of: 16.7 +/- 0.3 microM, 34.2 +/- 0.9 microM and 740 +/- 13 microM for niflumic acid, flufenamate and DPC respectively. The results indicate that at concentrations commonly used to block channel activity these chloride channel blockers have profound effects upon pHi.     

Inhibition of low Km cyclic GMP phosphodiesterases and potentiation of guanylate cyclase activators by cicletanine.

Cicletanine is an antihypertensive/vasorelaxant/natriuretic agent of unknown mechanism. We wished (a) to determine if cicletanine interacts with guanylate cyclase activators that modulate vasomotor tone and sodium balance [i.e., atriopeptin II (AP II), endothelium-derived relaxing factor (EDRF), and sodium nitroprusside (SNP)], and (b) to define the subcellular basis for this interaction by quantitating the effects of cicletanine on low Km cyclic GMP phosphodiesterase (PDE) activity. In phenylephrine-contracted rat aortic smooth muscle, the vasorelaxant potency of cicletanine was increased twofold in the presence of a threshold-relaxant concentration of AP II, and functional cyclic GMP PDE inhibition was also evident from the three- to sixfold potentiation by cicletanine of AP II- or SNP-induced vasorelaxation. Vasorelaxation produced by cicletanine was not endothelium dependent, however. In further studies, intravenous (i.v.) administration of cicletanine or the low Km cyclic GMP PDE inhibitor, zaprinast, decreased blood pressure (BP) greater than or equal to 20% in conscious spontaneously hypertensive rats (SHR). These results are consistent with the additional finding that cicletanine inhibited Ca2(+)-calmodulin (CaM) cyclic GMP PDE and zaprinast-sensitive cyclic GMP specific PDE over a concentration range (10-600 microM) similar to that for vasorelaxation. Thus, inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant effect of cicletanine as well as the potentiation by cicletanine of the vasorelaxant actions of guanylate cyclase activators. The extent to which this mechanism contributes to the antihypertensive efficacy of cicletanine has not yet been fully determined.     

Effects of arginine derivatives on soluble guanylate cyclase from neuroblastoma N1E 115 cells

The effects of L-arginine (Arg) derivatives on soluble guanylate cyclase from neuroblastoma N1E 115 cells were examined. The Arg derivatives were modified at the -NH2, -COOH, C alpha-proton or guanidino group of Arg. Among the synthesized derivatives, eight compounds, i.e. the 5-(dimethylamino)-1-naphthalenesulfonyl (DNS) ones, especially N-cyclohexyl-2-(N-DNSamino)-5-guanidino-2-methylvaleramide and 1-[2-(N-DNSamino)-2-(2-imino-1,2,3,4,5,6-hexahydropyrimidin- 4-yl)acetyl]- piperidine, were found to inhibit the activity of crude guanylate cyclase in the 105,000 g supernatant fraction of the cell homogenate. The enzyme, partially purified by a column of Chelex 100 Na+, was also inhibited by these eight compounds. The mode of the inhibition was competitive. The Ki values were in the range of 2-8 microM for the enzyme in the 105,000 g supernatant fraction and 3-16 microM for the partially purified enzyme, in the presence of Mg2+ as a metal cofactor. In contrast, a new derivative, methyl 2-amino-5-guanidinovalerate (M Arg ME), as well as the Arg methyl ester (Arg ME) and Arg; were found to enhance the activity of the partially purified guanylate cyclase; KA values of M Arg ME, Arg ME and Arg were approximately 9, 4 and 3 microM respectively. From these results, the free guanidino group including 2-imino-1,2,3,4,5,6-hexahydropyrimidin-4-yl or 2-imino-1,2,3,4,5,6-hexahydropyrimidin-5-yl and modification of the --NH2 residue with a hydrophobic group such as DNS seemed to be essential for inhibition of the guanylate cyclase; however, the guanidino and --NH2 residue of Arg should be free for activation by these Arg derivatives.     

Stimulation of soluble coronary arterial guanylate cyclase by SIN-1

SIN-1, a metabolite of the vasodilating drug molsidomine, was found to stimulate dose dependently (0.01-1 mM) soluble guanylate cyclase from bovine coronary arteries up to 100-fold the control value. The stimulatory effect of SIN-1 increased with rising concentrations of MnC1(2) or MgC1(2) and was diminished in the presence of methylene blue or ferricyanide. The time course of SIN-1-induced guanylate cyclase stimulation was characterized by a lag phase which was not observed after preincubation of the enzyme with SIN-1. In contrast to nitroglycerin and sodium nitroprusside, SIN-1 did not require the presence of cysteine or other thiols to stimulate guanylate cyclase. The results presented in this study provide further evidence that SIN-1 exerts its dilating effect on coronary vessels via direct stimulation of guanylate cyclase.     

Effect of brain natriuretic peptide on cyclic GMP accumulation in a kidney epithelial cell line (LLC-PK1)

The effect of porcine brain natriuretic peptide (pBNP) on cyclic GMP accumulation was studied in the kidney epithelial cell line, LLC-PK1. The addition of pBNP to the LLC-PK1 cells produced a time- and concentration-dependent increase in cyclic GMP accumulation and this effect was equipotent to that of alpha-human atrial natriuretic peptide (alpha-hANP). The simultaneous addition of pBNP and alpha-hANP at the maximal effective concentration of 10(-6) M did not have an additive effect on the cyclic GMP contents. The findings suggest that pBNP and alpha-hANP may share the same receptor in the LLC-PK1 cells.     

Effects of lipoproteins on cyclosporine A toxicity and uptake in LLC-PK1 pig kidney cells.

Cyclosporine A (CSA) is an effective immunosuppressant, but side effects such as renal toxicity can limit its therapeutic use. The current studies investigate the effects of lipoproteins on CSA-induced renal toxicity in the pig epithelial cell line LLC-PK(1). Protein synthesis and tritiated CSA were used as measures of toxicity and uptake of CSA, respectively, in the LLC-PK(1) cell line. The three main classes of lipoproteins, very low (VLDL), low (LDL), and high density lipoproteins (HDL) at hypo-, normo-, and hyperlipidemic levels were tested for their ability to affect CSA-induced toxicity and uptake. The major component of each lipoprotein was also tested to determine its effects on CSA-induced toxicity and uptake. ApoA-I, the major protein component of HDL, and intact LDL particles showed the most significant effects of CSA uptake and toxicity. The uptake and toxicity of CSA was effectively reduced with elevated LDL concentrations but showed a significant increase (p < 0.05) when incubated with elevated concentrations of apoA-I. Increasing VLDL and HDL concentrations slightly reduced CSA toxicity and uptake, but showed little effect with increased incubation time. Triglyceride and cholesterol, the respective major components of VLDL and LDL, did not alter CSA uptake or toxicity under the conditions tested. LDL and apoA-I are identified as the major effectors of CSA toxicity and uptake in LLC-PK(1) cells. These effects may be mediated through receptors such as the LDL receptor or those involved in protein reabsorption. The data presented here clearly demonstrate a relationship between CSA-induced toxicity and the nature of the associated lipoprotein.     

Dopamine synthesis and release in LLC-PK1 cells

A porcine-derived renal epithelial cell line, LLC-PK1, was used to examine dopamine (DA) synthesis from L-dopa and DA release into the media. DA synthesis and release were elevated by increasing either L-dopa concentration or incubation time. DA synthesis was shown to be entirely due to conversion of L-dopa to DA by aromatic amino acid decarboxylase. DA release from LLC-PK1 cells was stimulated by NaCl and other Na+ or Cl- salts. DA release by LLC-PK1 cells was not dependent on extracellular Ca2+ or significantly stimulated by the depolarizing actions of KCl. LLC-PK1 cells which are devoid of any neural contributions to renal DA production can synthesize DA from L-dopa and release DA in response to stimulation by either Na+ or Cl-.     

Secretion mode and subcellular localization of human interferon-beta exogenously expressed in porcine renal epithelial LLC-PK1 cells

We have investigated the secretion polarity of human interferon-beta (HuIFN-beta) exogenously expressed in the porcine renal proximal tubule cell line, LLC-PK1. In these cells, stably expressed HuIFN-beta was secreted to the apical and basolateral sides. However, when transiently expressed by apical lipofection, HuIFN-beta was secreted to both cell sides, while basolateral-preferential secretion was seen for basal transfection. Confocal imaging using enhanced green fluorescent protein (EGFP)-tagged HuIFN-beta revealed no difference in the subcellular distribution in either of the chimeric protein-expressing cells examined. These results suggest that the secretion polarity of HuIFN-beta is regulated by a post trans-Golgi network in a cell type-dependent manner.     

Contribution of reactive oxygen species to para-aminophenol toxicity in LLC-PK1 cells

para-aminophenol (PAP) causes nephrotoxicity by biochemical mechanisms that have not been fully elucidated. PAP can undergo enzymatic or non-enzymatic oxidation to form reactive intermediates. Using modulators of reactive oxygen species (ROS), the role of ROS in PAP toxicity in LLC-PK(1) cells was investigated. ROS formation was determined using a fluorescein derivative and viability using alamarBlue. Following treatment of cells with PAP, ROS formation occurred prior to loss of cell viability. Several modulators of ROS were used to identify the pathways involved in PAP toxicity. Viability was improved with catalase treatment, while viability was decreased when cells were treated with superoxide dismutase (SOD). Both catalase and SOD exert their effects outside of cells in the incubation medium, since there was no evidence of uptake of these enzymes in LLC-PK(1) cells. In cell-free incubations, hydrogen peroxide (H(2)O(2)) was produced when 0.5 mM PAP was included in the incubation medium. Further, SOD greatly increased and catalase greatly decreased H(2)O(2) production in these cell-free incubations. These data suggest that H(2)O(2) formed in the incubation medium contributes to loss of viability following PAP treatment. When cells were coincubated with 0.5 mM PAP and tiron, pyruvate, bathocuproine, 1, 10-phenanthroline, or dimethylthiourea (DMTU), ROS formation was decreased. However, there was minimal improvement in cell viability. Paradoxically, DMTU exacerbated PAP-induced loss of viability. These data suggest that ROS are generated in cells exposed to PAP but these species are not the predominant cause of cellular injury.     

Transcellular transport of creatinine in renal tubular epithelial cell line LLC-PK1

BACKGROUND/AIM: Creatinine is excreted into urine via tubular secretion in addition to glomerular filtration. In the present study, characteristics of the creatinine transport in renal epithelial cells were investigated. METHODS: The transcellular transport and accumulation of [14C]creatinine and [14C]tetraethylammonium (TEA) were assessed using LLC-PK1 cell monolayers cultured on porous membrane filters. RESULTS: [14C]Creatinine was transported directionally from the basolateral to apical side of LLC-PK1 cell monolayers. Basolateral uptake of [14C]creatinine was dependent on membrane potential, and was saturable with apparent K(m) and V(max) values of 13.2+/-2.8 mM and 13.1+/-3.1 nmol/mg protein/5 min, respectively. Concomitant administration of organic cations (1 mM) such as cimetidine, quinidine and trimethoprim inhibited both the transcellular transport and accumulation of [14C]creatinine. Furthermore, apical excretion of [14C]creatinine was not dependent on acidification of the apical medium. CONCLUSIONS: Creatinine was subjected to directional transport across renal epithelial cells from the basolateral to apical side. The organic cation transporter should be involved in the basolateral uptake of creatinine.     

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.