Involvement of guanylyl cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule

【摘要】  In killifish renal proximal tubules, endothelin-1 (ET-1), acting through a basolateral ET B receptor, nitric oxide synthase (NOS), and PKC, decreases cell-to-lumen organic anion transport mediated by the multidrug resistance protein isoform 2 (Mrp2). In the present study, we examined the roles of guanylyl cyclase and cGMP in ET signaling to Mrp2. Using confocal microscopy and quantitative image analysis to measure Mrp2-mediated transport of the fluorescent drug fluorescein methotrexate (FL-MTX), we found that oxadiazole quinoxalin (ODQ), an inhibitor of NO-sensitive guanylyl cyclase, blocked ET-1 signaling. ODQ was also effective when signaling was initiated by nephrotoxicants (gentamicin, amikacin, diatrizoate, HgCl 2, and CdCl 2 ), which appear to stimulate ET release from the tubules themselves. ODQ blocked the effects of the NO donor sodium nitroprusside but not of the phorbol ester that activates PKC. Exposing tubules to 8-bromo-cGMP (8-BrcGMP), a cell-permeable cGMP analog, decreased luminal FL-MTX accumulation. This effect was abolished by bisindoylmaleimide (BIM), a PKC inhibitor, but not by N G -methyl- L -arginine, a NOS inhibitor. Together, these data indicate that ET regulation of Mrp2 involves activation of guanylyl cyclase and generation of cGMP. Signaling by cGMP follows NO release and precedes PKC activation.

【关键词】  endothelin signaling protein kianse C xenobiotic transport

MULTIDRUG RESISTANCE PROTEINS (MRPs) are ATP-driven xenobiotic export pumps that belong to the ATP-binding cassette superfamily (ABC superfamily). These transporters were initially found to be expressed in tumor cell lines resistant to chemotherapeutics. However, they also contribute to important xenobiotic defense mechanisms in barrier and excretory tissues. P-glycoprotein ( ABCB1 ), breast cancer-related protein ( ABCG2 ) ( 16, 31 ), and members of the ABCC subfamily (the MRPs) limit xenobiotic absorption from the gut and xenobiotic entry into the central nervous system ( 3, 5, 20 ). They are also present in the liver and kidney, organs important for the excretion of potentially toxic xenobiotics, xenobiotic metabolites, and endogenous waste products ( 19, 26 ). The apical localization of P-glycoprotein and MRP2 in hepatocytes and renal proximal tubule epithelial cells is consistent with their importance in excretory transport into bile and urine. In addition, some MRPs may play a role in cellular signaling by transporting second messengers like cyclic nucleotides and leukotrienes ( 2, 13, 34 ).

We previously used intact killifish ( Fundulus heteroclitus ) renal proximal tubules to demonstrate that Mrp2 function is regulated by the vasoactive peptide endothelin (ET) working through a basolateral ET B receptor, nitric oxide synthase (NOS), nitric oxide (NO), and PKC ( 18, 23, 33 ). Firing this signaling pathway rapidly reduced transport mediated by Mrp2. Importantly, this autocrine/paracrine signaling pathway was also triggered by acute exposure to low levels of nephrotoxicants, which caused Ca-dependent release of ET ( 33 ).

The present study is concerned with the mechanism by which NO activates PKC in killifish renal proximal tubules. Specifically, we found that inhibition of guanylyl cyclase blocked ET signaling whether initiated by ET-1 or by nephrotoxicants. In addition, 8-bromo-cGMP (8-BrcGMP) reduced Mrp2-mediated transport, and this effect was blocked when PKC was inhibited but not when NOS was inhibited. Thus guanylyl cyclase appears to be involved in signaling by ET and the nephrotoxicants; generation of cGMP follows NO release and precedes PKC activation.

METHODS

Chemicals. Fluorescein methotrexate (FL-MTX), bisindolymaleimide (BIM), and N G -methyl- L -arginine acetate salt ( L -NMMA) were purchased from Molecular Probes (Eugene, OR). RES-701-1, an ET B -receptor antagonist, was obtained from Peninsula Laboratories (Belmont, CA). Sodium nitroprusside (SNP) and oxadiazole quinoxalin (ODQ) were purchased from Calbiochem (San Diego, CA). PMA was obtained from Alexis Biochemicals (San Diego, CA) and from Sigma (St. Louis, MO). 8-BrcGMP and its Rp isoform, HgCl 2, and CdCl 2, gentamicin, amikacin, and diatrizoic acid were purchased from Sigma. All other chemicals used were obtained at the highest purity available commercially.

Animals and tissue preparation. Killifish were collected by local fishermen in the vicinity of Mount Desert Island, ME, and maintained at the Mount Desert Island Biological Laboratory in tanks with natural flowing seawater. Renal tubular masses were isolated in a marine teleost saline based on that of Forster and Taggart ( 4 ) containing (in mM) 140 NaCl, 2.5 KCl, 1.5 CaCl 2, 1.0 MgCl 2, and 20 Tris at pH 8.0. All experiments were carried out at 18-20°C. Under a dissecting microscope, each mass was teased with fine forceps to remove adherent hematopoietic tissue. Individual killifish proximal tubules were dissected and transferred to a foil-covered Teflon chamber containing 1.5 ml of marine teleost saline with 1 µM FL-MTX and added effectors. The chamber floor was a 4 x 4-cm glass coverslip to which the tubules adhered lightly and through which the tissue could be viewed by means of an inverted microscope. Tubules were incubated at room temperature for 30 min until a steady state was reached for FL-MTX. Analysis of tubule extracts by HPLC showed no metabolic degradation of FL-MTX when incubated with killifish proximal tubules for periods of at least 1 h ( 17, 29 ).

Confocal microscopy. The chamber containing renal tubules was mounted on the stage of an Olympus FluoView inverted confocal laser-scanning microscope and viewed through a x 40 water-immersion objective (numerical aperture 1.15). Excitation was provided by the 488-nm line of an argon ion laser. A 510-nm dichroic filter and a 515-nm long-pass emission filter were used. Neutral-density filters and low laser intensity were used to avoid photobleaching. With the photomultiplier gain set to give an average luminal fluorescence intensity of 1,500-3,000 (on a scale of 0-4,096), tissue autofluorescence was undetectable. To obtain an image, dye-loaded tubules in the chamber were viewed under reduced, transmitted light illumination, and a single proximal tubule with well-defined lumen and undamaged epithelium was selected. The plane of focus was adjusted to cut through the center of the tubular lumen, and an image was acquired by averaging four scans. The confocal image was viewed on a high-resolution monitor and saved to an optical disk or a Zip disk. In previous studies, it has been shown that there is a linear relationship between fluorescence intensity and dye concentration ( 22 ). However, because of the many uncertainties in relating cellular fluorescence to actual compound concentration in cells and tissues with complex geometry, data are reported here as average measured pixel intensity rather than estimated dye concentration. Fluorescence intensities were measured from stored images using Scion image version 1.8 for Windows as described previously ( 17, 21 ). Briefly, two or three adjacent cellular and luminal areas were selected from each tubule, and the average pixel intensity for each area was calculated. The values used for that tubule were the means of all selected areas after subtraction of the pixel intensity of the bathing medium, which was considered as background.

Data analysis. Data are given as means ± SE. Mean values were considered to be significantly different when P < 0.05 by use of an unpaired t -test or one-way ANOVA followed by Bonferroni's multiple comparison test. Software used for statistical analysis was GraphPad Prism (version 3.00 for Windows; GraphPad Software, San Diego, CA).

RESULTS

The present experiments were conducted using isolated renal proximal tubules from a marine teleost fish, the killifish, to determine whether guanylyl cyclase and cGMP are involved in the regulation of Mrp2-mediated transport. This comparative animal model has proven to be a powerful tool for the study of secretory transport in an intact proximal tubule ( 24 ). As in mammalian proximal tubules, killifish express high levels of Mrp2 in the luminal membrane of renal proximal tubule cells. Moreover, intact killifish tubules exhibit Mrp2-mediated transport of a number of fluorescent substrates, e.g., FL-MTX, that can be visualized and measured using confocal microscopy ( 17, 18, 21 ). Figure 1 A shows a typical confocal image of a control killifish tubule after 30-min (steady state) incubation in medium with 1 µM FL-MTX. Autofluorescence was not detectable. The fluorescence distribution pattern is the same as shown previously, medium ( 17, 18 ). This pattern is indicative of a two-step process, involving uptake at the basolateral membrane mediated by an as yet uncharacterized transporter for large organic anions and secretion into the lumen mediated by a teleost form of Mrp2 (for data on substrate and inhibitor specificities as well as immunostaining with Mrp2 antibodies, see Refs. 18 and 33 ). Using an Sf9 overexpression system, we previously proved that FL-MTX is a substrate for MRP2 ( 32 ). Interference of other members of the Mrp family with FL-MTX transport in this model is unlikely, although other Mrps are known to share numerous substrates. However, Mrp5 and Mrp6 are located in the basolateral membrane and not in the apical membrane of renal proximal tubules, whereas Mrp1 and Mrp3 are not expressed in renal proximal tubules ( 26 ). Furthermore, we can exclude the contribution of Mrp4 because preliminary results from our group show that FL-MTX is not a substrate for MRP4 (Smeets PH and Russel FGM, unpublished observations), and MRP4-mediated transport is insensitive to leukotriene C 4 ( 34 ), which is an excellent inhibitor of FL-MTX secretion in killifish proximal tubules ( 17, 18 ).

Fig. 1. Representative confocal images of killifish proximal tubules after incubation in marine teleost saline with 1 µM fluorescein methotrexate (FL-MTX) for 30 min in the absence ( A ) and presence of the cGMP analog 8-bromo-cGMP (8-BrcGMP; B ). Treatment with 1 µM 8-BrcGMP reduced luminal fluorescence, indicating that FL-MTX secretion on multidrug resistance protein isoform 2 (Mrp2) was inhibited.

To determine whether ET signaling involved activation of guanylyl cyclase, we examined the effects of the NO-sensitive guanylyl cyclase inhibitor ODQ on FL-MTX transport and its modulation by ET-1. Figure 2 shows that exposure to 10 nM ET-1 resulted in a decrease in luminal accumulation of FL-MTX by 50% and an unchanged cellular accumulation, a result consistent with previous experiments ( 18 ). This inhibition pattern is consistent with that observed in earlier experiments after exposure to specific Mrp2 inhibitors such as leukotriene C 4 ( 17, 18 ) and taken to mean that FL-MTX efflux into the lumen is not an important determinant of steady-state cellular FL-MTX accumulation. Indeed, time course experiments showed a rapid increase in cellular and luminal fluorescence in control tubules that reached a steady state after 10 min. For tubules exposed to 10 nM ET-1 from time 0 on, cellular fluorescence approximated control values, but luminal fluorescence was significantly lower than controls except at the earliest time measured ( 18 ). This indicates that the steady-state cellular levels of FL-MTX are set independently of events at the luminal membrane. Exposing tubules to 10 µM ODQ by itself did not affect luminal FL-MTX accumulation and thus transport. When tubules were exposed to ET-1 plus ODQ, no significant reduction in luminal accumulation of FL-MTX was found. None of these treatments affected cellular FL-MTX accumulation, again indicating that steady-state cellular levels of FL-MTX seem to set independently of events at the luminal membrane. Since we previously demonstrated that several nephrotoxicants also fired ET signaling in the tubules ( 33 ), we also determined whether nephrotoxicant effects on signaling and FL-MTX transport could be blunted by ODQ. Consistent with previous results ( 23, 33 ), exposing tubules to gentamicin, amikacin, diatrizoate, HgCl 2, and CdCl 2 significantly reduced luminal accumulation of FL-MTX ( Table 1 ); cellular accumulation was not affected (not shown). The concentrations of nephrotoxicants used here do not reduce transport of FL by the classic Na-dependent organic anion system ( 33 ) and do not reduce mitochondrial membrane potential measured using a fluorescent indicator dye (Notenboom S, Miller DS, Russel FGM, and Masereeuw R, unpublished observations). Importantly, when tubules were pretreated with 10 µM ODQ, none of the nephrotoxicants significantly reduced luminal FL-MTX accumulation. Thus inhibiting guanylyl cyclase blocked signaling through the ET B receptor-NOS-PKC pathway irrespective of whether the stimulus was a hormone or nephrotoxicant.

Fig. 2. Prevention of the inhibitory effect of endothelin-1 (ET-1) on FL-MTX transport by the guanylyl cyclase inhibitor oxadiazole quinoxalin (ODQ). The inhibitory effect caused by 10 nM ET-1 on FL-MTX transport is prevented by 10 µM ODQ, indicating cGMP generation after ET-1 release. Values are means ± SE for 10-15 tubules from 1 fish. ***Significantly lower than the control value ( P < 0.001).

Table 1. Inhibition of FL-MTX transport by nephrotoxicants and protection by 10 µM ODQ

We used ODQ as a pharmacological tool to determine the position of guanylyl cyclase in the signaling chain. Figure 3 shows that ODQ attenuates the reduction in luminal FL-MTX accumulation caused by the NO donor SNP but has no effect on the reduction caused by the PKC activator PMA. Thus activation of guanylyl cyclase appears to follow NO release and precedes PKC activation.

Fig. 3. Effects of guanylyl cyclase inhibitor ODQ on FL-MTX transport inhibited by nitric oxide (NO) and PKC stimulation. The inhibitory effect caused by 100 µM sodium nitroprusside (SNP) on FL-MTX transport is prevented by 10 µM ODQ ( A ). However, the inhibitory effect caused by 100 nM PMA on FL-MTX transport could not be prevented by 10 µM ODQ ( B ). Together, these data indicate that cGMP generation follows NO release and precedes PKC stimulation. Values are means ± SE for 10-14 tubules from 1 fish. ***Significantly lower than the control value ( P < 0.001).

Next, we examined the effects of cGMP analogs on FL-MTX transport. Figure 1 B shows that incubating tubules in medium with 1 µM 8-BrcGMP (a membrane-permeant analog that activates PKG) reduced luminal but not cellular FL-MTX accumulation. Quantitation of images indicated that the reduction in luminal accumulation was concentration dependent, with a significant decrease seen 50% decrease with 1 µM ( Fig. 4 A ). Rp-8-BrcGMP, which does not activate PKG, also reduced luminal accumulation of FL-MTX ( Fig. 4 B ) but was less effective.

Fig. 4. Dose-dependent inhibitory effect of 8-BrcGMP ( A ) and its inactive Rp isoform (Rp-8-BrcGMP; B ) on FL-MTX transport. Values are means ± SE for 15-19 tubules from 1 fish. *Significantly lower than the control value ( P < 0.05). ***Significantly lower than the control value ( P < 0.001).

If 8-BrcGMP reduced Mrp2-mediated transport of FL-MTX through signaling rather than by competing for transport, its effects should be attenuated when the signaling chain is broken by inhibiting a downstream step, i.e., PKC activation. Figure 5 shows a series of experiments designed to test this possibility. First, inhibition of PKC by BIM abolished the effects of 1 µM 8-BrcGMP on luminal FL-MTX accumulation ( Fig. 5 A ). Second, inhibition of NOS by L -NMMA did not alter the effects of 8-BrcGMP ( Fig. 5 B ). These results are consistent with 8-BrcGMP modifying transport through signaling at a step downstream of NOS but upstream of PKC. Third, in contrast, BIM exposure did not alter the effects of Rp-8-BrcGMP ( Fig. 5 A ), suggesting that this compound reduced transport by interacting with Mrp2. However, pilot experiments using MRP2-transfected Sf9 vesicles, as previously described by Terlouw et al. ( 32 ), showed no inhibition of FL-MTX transport (in pmol FL-MTX·mg protein -1 ·min -1 ) by 10 µM 8-BrcGMP (82.1 ± 11.3; n = 3), 100 µM 8-BrcGMP (186 ± 47.6; n = 3), 10 µM Rp-8-BrcGMP (147 ± 32.5; n = 3), 100 µM 8-BrcGMP (165 ± 16.4; n = 3) compared with control (91.7 ± 46.3; n = 3) (data not shown).

Fig. 5. Consequences of PKC and NO synthase (NOS) inhibition on FL-MTX transport reduced by cGMP. The inhibitory effect caused by 1 µM 8-BrcGMP, but not by its inactive Rp isoform, on FL-MTX transport is prevented by 100 nM bisindoylmaleimide (BIM; A ). The inhibitory effect caused by 1 µM 8-BrcGMP could not be prevented by 50 µM N G -methyl- L -arginine ( L -NMMA; B ). Together, these data indicate that cGMP generation precedes PKC stimulation and follows NO release. Values are means ± SE for 9-19 tubules from 1 fish. *Significantly lower than the control value ( P < 0.05). ***Significantly lower than the control value ( P < 0.001).

DISCUSSION

cGMP is an intracellular second messenger involved in hormonal signaling throughout the body. cGMP is generated from GTP by guanylyl cyclases, which are present as membrane-bound and soluble forms ( 1 ). The soluble forms produce cGMP in response to several signals, including NO ( 25, 36 ). cGMP itself acts through cGMP-dependent PKG, cyclic nucleotide-gated channels, cAMP-dependent protein kinase, and phosphodiesterase ( 15 ). Here, we provide evidence that NO-dependent guanylyl cyclase and cGMP are involved in the regulation of Mrp2-mediated transport in the renal proximal tubule. We previously showed that ET, acting through a basolateral ET B receptor, NOS, and PKC, decreases cell-to-lumen organic anion transport mediated by Mrp2 ( 18, 23 ). Figure 6 summarizes this sequence of events. Transport is also reduced by several nephrotoxicants, which cause Ca-dependent ET release from the tubules; ET then activates signaling by an autocrine/paracrine mechanism ( 33 ).

Fig. 6. Scheme illustrating the proposed sequence of events by which nephrotoxicants reduce Mrp2-mediated transport in isolated renal proximal tubules. Nephrotoxicants cause a transient opening of the calcium channels, which increases intracellular calcium concentration and stimulates ET release. The hormone binds to a basolateral ET B receptor, which activates NOS, increases NO production, activates soluble guanylyl cyclase (sGC), increases cGMP production, and activates PKC. PKC activation rapidly reduces transport by Mrp2.

The present study shows that ODQ, an inhibitor of guanylyl cyclase, blocked ET signaling to Mrp2. ODQ was effective irrespective of the source of the initial signal, i.e, hormone or nephrotoxicant. ODQ also blocked the effects of the NO generation by SNP but not the effects of PKC activation by PMA. 8-BrcGMP reduced Mrp2-mediated transport, and this effect was blocked by PKC inhibition, but not by NOS inhibition. Although the Rp-isoform of 8-BrcGMP, which does not activate PKG, also reduced transport on Mrp2, this effect was not altered by BIM. Thus it is likely that the Rp isoform, unlike the parent compound, affected transport by interacting directly with the transporter. Together, the data indicate that ET signaling involves activation of guanylyl cyclase and generation of cGMP. This step in signaling occurs after NO generation by NOS and before PKC activation.

Although our results indicated that cGMP activates PKC, other protein kinases may still be involved as intermediate steps. Possible candidates are PKA and PKG. However, PKA activation does not appear to be involved, because we previously found no effect of a PKA-selective inhibitor on ET-1 signaling ( 18 ). Our attempts to demonstrate activation of PKG as an intermediate step were not successful, because each of the several PKG-selective inhibitors tested inhibited transport on Mrp2 themselves (Notenboom S and Miller DS, unpublished observations). It is likely that these drugs affected transport by direct interaction with the transporter, because all inhibitors were organic anions. Additional experiments will be needed to clarify at the molecular level the events between cGMP production and PKC activation and the events between PKC and Mrp2 inhibition. A possible candidate for the latter is phosphorylation of Mrp2 PSD-95/Disc-large/zona occludin-1 (PDZ) domains. Hegedüs et al. ( 7 ) suggested that PKC is involved in MRP2 targeting and recycling through phosphorylation of the MRP2 PDZ domain, which influences the interaction between MRP2 and its anchoring PDZ proteins and thereby its transport function.

Signaling through cGMP has been implicated in the mechanisms of action of several nephrotoxicants. Tack et al. ( 30 ) showed that a single dose of cyclosporine A transiently increases glomerular cGMP in rats. In this process, activation of ET B receptors and the NO pathway is also involved. Signaling through NO and cGMP also affects renal tubular transport, e.g., sodium transport in rabbit proximal tubule ( 25 ) and Na + -K + -ATPase in rat proximal tubule ( 36 ). cGMP has been implicated in the regulation of other transport processes in the kidney, although not directly linked to NO production or to PKC stimulation. For example, Hirsch et al. (8-10) described that cGMP is involved in the regulation of Ca 2+ and K + channels in a human proximal tubule cell line, whereas others ( 28 ) showed that cGMP is involved in the regulation of the organic cation transporters, rOCT1 and hOCT2. cGMP has also been found to be involved in the regulation of vascular tone in which the ET B receptor is included. Here also, binding of ET-1 to the ET B receptor leads to the production of NO and subsequent cGMP. However, cGMP, in turn, inhibits ET-1 release, suggesting a complex signaling mechanism for relaxation of pulmonary vessels ( 14 ). A comparable negative-feedback system between cGMP and ET-1 secretion is not present in our system, because the inhibitory effect of cGMP could be completely prevented by the PKC inhibitor BIM. A role for cGMP in renal toxicity is not yet established. It was shown previously that cytotoxicity of oxidant stress resulted in upregulation of NOS with excessive production of NO ( 6 ). The nephrotoxicants cyclosporin A, FK506 ( 11 ), and the heavy metal HgCl 2 ( 35 ) have all been implicated in acute renal failure through increased NO production. The biological actions of NO are mediated often by cGMP. Hosogai et al. ( 12 ) found that exposure to cyclosporin A resulted in a decrease in cGMP-phosphodiesterase activity and an increase in guanylate cyclase activity, implying a role for cGMP in cyclosporine A-induced nephrotoxicity. Next to the activity of the soluble guanylyl cyclases and phosphodiesterases, excretion and reabsorption of cGMP in the proximal tubule might influence transport processes important for fluid balance and possibly for Mrp2 regulation ( 13, 27, 34 ).

In summary, cGMP plays a role in the short-term regulation of Mrp2 by the following sequence of events: nephrotoxicants trigger a Ca influx, ET is released and binds to the ET B receptor, the ET B receptor triggers NO release by activating NOS, subsequently soluble guanylyl cyclase is activated, and the cGMP produced stimulates PKC, eventually leading to the inhibition of Mrp2-mediated transport. In conclusion, cGMP plays a role in the ET-signaling pathway, first described by Masereeuw et al. ( 18 ) in killifish proximal tubule, next to its diverse actions throughout the body.

GRANTS

This study was supported by the Dutch Kidney Foundation and National Institute of Environmental Health Sciences Grant ES-03828.

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作者单位：1 Department of Pharmacology and Toxicology, University Medical Center Nijmegen, Nijmegen Center for Molecular Life Sciences, 6500 HB Nijmegen, The Netherlands; 3 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Science, National Institutes of Health, Research Tri