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作者：Ping Li, Houli Jiang, LiMing Yang, Shuo Quan, Sandra Dinocca, Francisca Rodriguez, Nader G. Abraham, and Alberto Nasjletti作者单位：Department of Pharmacology, New York Medical College, Valhalla, New York 10595
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【摘要】* s* h1 M0 l/ H4 h% G& T. m i7 U
Heme oxygenase (HO)-derived carbon monoxide (CO) attenuates vascular reactivity to constrictor stimuli. ANG II produces vasoconstriction and induces HO-1 isoform expression. However, direct evidence that ANG II promotes HO product generation is lacking. Therefore, we examined the effects of ANG II on CO release and HO isoform expression in isolated rat kidneys. Kidneys were perfused with oxygenated Krebs buffer. ANG II (1 µmol/l) increased ( P < 0.05) perfusion pressure from 97 ± 9 to 150 ± 14 mmHg; it also increased ( P < 0.05) the concentration of CO in the venous effluent (from 27.1 ± 11.9 to 45.6 ± 11.7, 62.5 ± 16.7, 94.8 ± 20.7, and 101.9 ± 13.1 nmol/l after 30, 60, 90, and 120 min, respectively). The pressor effect of ANG II was blunted ( P < 0.05) in kidneys perfused with buffer containing losartan (10 µmol/l) or PKC inhibitors staurosporine (0.1 µmol/l) or calphostin C (1 µmol/l). Kidneys perfused with buffer containing ANG II for 120 min also displayed increased ( P < 0.05) HO-1 expression. Stannous mesoporphyrin (30 µmol/l) decreased CO release ( P < 0.05) in preparations perfused with and without ANG II; the HO inhibitor also increased ( P < 0.05) perfusion pressure, more so in kidneys perfused with that without ANG II. We conclude that ANG II stimulates CO production and release in isolated, perfused rat kidneys. This action of ANG II is linked to the activation of AT 1 receptors and involves PKC activation and upregulation of renal HO-1 but not of HO-2 protein expression. The study suggests upregulation of renal HO-1 and CO release are protagonic events in a counterregulatory mechanism that attenuates ANG II-induced renal vasoconstriction.
7 G0 l6 Q2 H* Y0 G! Y7 x/ F/ s0 z/ b 【关键词】 heme oxygenase renal hemodynamics vasoconstriction
4 w0 _' q0 Z1 E1 o) B HEME OXYGENASE (HO) ISOFORMS 1 (HO-1) and 2 (HO-2) catalyze the metabolism of heme to biliverdin, ferrous iron, and carbon monoxide (CO) ( 1, 20 ). In the kidney, both vascular and tubular structures express HO-1 ( 5, 12 ). The level of expression is low under basal conditions but increases greatly in response to various kinds of injury ( 5, 12 ). Overexpression of HO-1 is believed to ameliorate the renal damage produced by injurious conditions, presumably via a mechanism(s) involving reduction of cellular heme and/or augmented generation of the HO products CO and biliverdin ( 2, 6, 21, 23, 24 ).8 q, r8 X9 m, b- ]$ f
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Constitutive expression of HO-2 has been documented in renal vascular and tubular structures ( 5, 12, 15, 18 ). The catalytic activity of this enzyme increases as a result of PKC-mediated phosphorylation ( 8 ). CO derived from heme metabolism by renal HO-2 was reported to influence transport in the thick ascending limb of the loop of Henle ( 18 ) and vascular reactivity and tone in preglomerular vessels ( 15, 27 ).
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- y5 ~9 V# T$ HSeveral studies have identified areas of interaction between the heme-HO and the renin-angiotensin systems. Treatment with an inhibitor of HO intensifies the renal vasoconstrictor effect of ANG II ( 27 ) and aggravates the renal dysfunction that accompanies the development of severe ANG II-induced hypertension in rats ( 3 ). These observations imply that the renal HO system subserves a protective role that mitigates the injurious actions of ANG II. ANG II induces HO-1 expression in kidney and arterial vessels ( 3, 7, 11, 14 ). ANG II also may be expected to upregulate HO-2 activity as it is known to stimulate PKC signaling ( 8, 10 ). However, direct evidence that ANG II promotes HO product generation in intact tissues is lacking. Therefore, we conducted studies in isolated, perfused rat kidneys to investigate whether ANG II promotes HO-dependent generation of CO. We also examined the relationship between ANG II-induced CO release and the renal expression of HO isoforms, the involvement of PKC in the stimulatory action of ANG II on CO release, and the renal vascular response to HO inhibition in kidneys perfused with media containing and not containing ANG II.# j8 f+ T8 P& J+ G9 `8 e% o7 e
^. \, K# M) {METHODS
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Drugs and solutions. Stannous mesoporphyrin (SnMP) was purchased from Frontier Scientific (Logan, UT); it was dissolved in 50 mmol/l Na 2 CO 3, sonicated, filtered, and further diluted in Krebs bicarbonate buffer immediately before use. All other drugs were obtained from Sigma (St. Louis, MO). Stock solutions of isoleucine 5 -ANG II were prepared in 0.01 mol/l acetic acid, and of phenylephrine in 0.15 mol/l NaCl. Stock solutions of PMA, staurosporine and calphostin C were prepared in DMSO; these solutions were diluted at least 4,000-fold with Krebs buffer during experimentation. The composition of Krebs bicarbonate buffer (mmol/l) was 118.5 NaCl, 4.7 KCl, 2.8 CaCl 2, 1.2 KH 2 PO 4, 1.1 MgSO 4, 25.0 NaHCO 3, and 11.1 dextrose.: j+ i; l5 }( v N. t
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Experimental procedures and protocols. Experiments were conducted in male Sprague-Dawley rats (300-400 g, Charles River, Wilmington, DE) in accordance with protocols approved by the Institutional Animal Care and Use Committee. The right kidney of rats anesthetized with pentobarbital sodium (50 mg/kg ip) was exposed by midline laparotomy, and the renal artery was cannulated with a 21-gauge needle connected to Sylastic tubing and the renal vein with polyethylene tubing (PE-90). Perfusion of the kidney was initiated immediately thereafter with gassed (95% O 2 -5% CO 2 ) Krebs bicarbonate buffer (37°C) delivered at 7-8 ml/min by means of a Masterflex pump (model 7520-00, Cole-Parmer Instrument, Barrington, IL). The renal venous effluent was not recirculated; it was sampled (1 ml) before and during experimental interventions by puncturing the venous cannula with a needle connected to a gas-tight syringe and analyzed for CO. Perfusion pressure was monitored continuously using a pressure transducer (model P23ID, Statham Division, Gould, Oxnard, CA) coupled to a polygraph (model 7D, Grass Instruments, Quincy, MA). Experimental protocols were implemented after a 30- to 40-min equilibration interval.
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Protocol 1 was designed to investigate the effect of ANG II and PKC activation on CO concentration in the renal venous effluent and the renal expression of HO-1 and HO-2. It included 11 groups in which data were collected during a control (30 min) and an experimental (120 min) period. Group 1 consisted of kidneys ( n = 6) perfused with Krebs buffer only during the control and experimental periods. Groups 2 ( n = 6), 3 ( n = 4), 4 ( n = 6), and 5 ( n = 4) consisted of kidneys perfused with Krebs buffer during the control period and Krebs buffer containing 1 µmol/l ANG II, 0.01 µmol/l ANG II, the PKC activator PMA (0.2 µmol/l), or phenylephrine (5.0 µmol/l) during the experimental period, respectively. Group 6 included kidneys ( n = 6) perfused with Krebs buffer containing the ANG II type 1 (AT 1 ) receptor-antagonist losartan (10 µmol/l) during the control period and with Krebs buffer containing both losartan (10 µmol/l) and ANG II (1.0 µmol/l) during the experimental period. Groups 7 ( n = 6) and 8 ( n = 6) consisted of kidneys perfused with Krebs buffer containing the PKC inhibitor staurosporine (0.1 µmol/l) during the control period and, respectively, staurosporine (0.1 µmol/l) plus ANG II (1.0 µmol/l) or plus PMA (0.2 µmol/l) during the experimental periods. Groups 9 ( n = 3) and 10 ( n = 3) consisted of kidneys perfused with Krebs buffer containing the PKC inhibitor calphostin C (1 µmol/l) during the control period and, respectively, calphostin C (1 µmol/l) plus ANG II (1 µmol/l) or plus PMA (0.2 µmol/l) during experimental periods. Group 11 ( n = 4) consisted of kidneys perfused with Krebs buffer containing the HO inhibitor SnMP (30 µmol/l) during the control period and SnMP (30 µmol/l) plus ANG II (1 µmol/l) during the experimental period. At the completion of the 120-min experimental period, kidneys from groups 1, 2, and 4 were used for measurement of HO isoform expression. In a complementary study, HO isoforms expression was assessed after a 30-min experimental period during which the kidneys were perfused as described for groups 1, 2, and 4 with Krebs buffer only ( n = 4), buffer containing ANG II (1 µmol/l; n = 4), and buffer containing PMA (0.2 µmol/l; n = 4), respectively.& \2 E6 K# r' j+ n/ L5 z0 G
9 n2 S2 K( K, I' W& [' F# jProtocol 2 was designed to contrast the effects of the HO inhibitor ( 9 ) SnMP on perfusion pressure and CO concentration in the venous effluent of kidneys perfused for 120 min with Krebs buffer containing ( n = 6) and not containing ( n = 6) ANG II (1 µmol/l). SnMP (30 µmol/l) was introduced into the perfusion buffer after a 60-min control period, and the kidneys were perfused for an additional 60 min.
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Analysis of CO in renal venous effluent. The renal venous effluent was sampled with a gas-tight syringe; a 1-ml specimen was immediately injected into an amber vial (2 ml) capped with rubberized Teflon liners. Subsequently, internal standards made of isotopically labeled CO ( 13 C 16 O and 13 C 18 O) were injected into the vials, and the CO content of the headspace gas was determined by gas chromatography/mass spectroscopy using an HP-5989A mass spectrometer interfaced to an HP-5890 gas chromatograph ( 15 ). The separation of CO from other gases was carried out on a GS-Molesieve capillary column (30 m, 0.53-mm internal diameter; J&W Scientific; Folsom, CA) kept at 40°C. Helium was used as the carrier gas with a linear velocity of 0.3 m/s. CO eluted at 3.6 min and was fully separated from N 2, O 2, H 2 O, and CO 2. The mass spectrometer parameters were 120°C ion source temperature, 31-eV electron energy, and 120°C transfer line temperature. Aliquots (100 µl) of the headspace gas of either standard solutions or experimental samples were injected using a gas-tight syringe into the spitless injector having a temperature of 120°C. Abundance of ions at 28, 29, and 31 mass-to-charge ratio ( m / z ) corresponding to 12 C 16 O, 13 C 16 O, and 13 C 18 O, respectively, was acquired via selected ion monitoring. The amount of CO in samples was calculated from standard curves constructed with abundance of ions at 28, 29, or 31 m / z. Both standard curves were linear over the range of 0.01-5.0 µmol/l, and both yielded comparable results when used for determining the concentration of endogenous CO. The sensitivity of the assay is 1 pmol CO. The results were expressed as nanomoles of CO per liter of renal venous effluent.
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Assessment of HO isoform expression. Perfused kidneys from groups 1-3 in protocol 1 were homogenized in ice-cold 50 mmol/l Tris·HCl buffer, pH 7.4, containing 1.0 mmol/l EDTA, 1% NP-40, 0.25% sodium deoxycholate, and 10% protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 10,000 g for 30 min. The supernatant was assayed for protein with a kit (Bio-Rad Laboratories, Hercules, CA) and saved for Western blot analysis of HO-1 and HO-2 according to published procedures ( 5 ). Briefly, 20 µg of 10,000- g supernatant protein were subjected to electrophoresis on SDS-12% polyacrylamide gels and then transferred to a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 5% bovine serum albumin-3% powdered milk in Tris-saline buffer (20 mmol/l Tris·HCl, pH 7.5, containing 150 mmol/l NaCl). Subsequently, the membrane was washed with Tris-saline buffer and then incubated with HO-1 or HO-2 antibodies (Stressgen, Victoria, BC) for 1 h at room temperature. The membrane was washed again with Tris-saline buffer before incubation at room temperature for 60 min with the secondary antibody (goat anti-rabbit immunoglobulins) conjugated to horseradish peroxidase (Bio-Rad Laboratories). After the final washes, the immunocomplexed bands were visualized with the chemiluminescence enhanced system (Amersham, Arlington Heights, IL) and quantified by densitometric analysis. t/ B, R! P1 _/ I9 f* k
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Statistics. Data are expressed as means ± SE. Data were analyzed by one- or two-way ANOVA for repeated measures. If differences were noted, the Newman-Keuls test was used to make specific comparisons. The null hypothesis was rejected when the probability value was 9 U$ U8 p- \0 U% q" `; k% f
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Figure 1 shows data on renal perfusion pressure and CO concentration in the venous effluent of isolated kidneys before and during perfusion with buffer containing 1 µmol/l ANG II. In kidneys perfused with Krebs buffer only during both the control and experimental periods, perfusion pressure and the concentration of CO were stable throughout the experiment, ranging between 88 ± 6 and 97 ± 6 mmHg and 7.0 ± 1.0 and 12.0 ± 2.5 nmol/l, respectively. The inclusion of 1 µmol/l ANG II into the Krebs buffer increased ( P
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Fig. 1. Perfusion pressure (PP) and concentration of carbon monoxide (CO) in the venous effluent of rat kidneys perfused with oxygenated Krebs bicarbonate buffer only ( n = 6), with buffer containing ANG II (1 µmol/l; n = 6), ANG II plus losartan (10 µmol/l; n = 6), or ANG II plus staurosporine (0.1 µmol/l; n = 6). ANG II was added to the perfusion buffer at time 0; losartan and staurosporine were introduced in the perfusion buffer 45 min before the addition of ANG II. Values are means ± SE. * P
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In control experiments, neither perfusion pressure (90 ± 9 vs. 93 ± 7 mmHg) nor the concentration of CO in the venous effluent (18.7 ± 4.0 vs. 19.3 ± 5.2 nmol/l) was altered in kidneys ( n = 3) perfused for 30 min with Krebs buffer containing 0.05% DMSO only, the solvent used to dissolve staurosporine and calphostin C. Moreover, in these kidneys subsequent perfusion for 60 min with buffer containing 1 µmol/l ANG II (along with 0.05% DMSO) still caused an elevation ( P
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Figure 2 illustrates the effect of the PKC activator PMA (0.2 µmol/l) on perfusion pressure and CO concentration in the venous effluent of kidneys perfused with Krebs buffer containing and not containing staurosporine (0.1 µmol/l). In kidneys perfused without the PKC inhibitor, PMA elicited a sharp and sustained increase ( P ! F" N9 j! @8 j% W' O) d
% [" W4 i# r! N" \) Z: cFig. 2. PP and concentration of CO in the venous effluent of rat kidneys perfused with oxygenated Krebs bicarbonate buffer containing PMA (0.2 µmol/l; n = 6) or PMA plus staurosporine (0.1 µmol/l; n = 6). PMA was added to the perfusion buffer at time 0; staurosporine was introduced into the buffer 45 min before the addition of PMA. Values are means ± SE. * P
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Figures 3 and 4 contrast data on the expression of HO-1 and HO-2 in kidneys perfused for 30 and 120 min, respectively, with Krebs buffer containing and not containing ANG II (1 µmol/l) or PMA (0.2 µmol/l). Relative to data in kidneys perfused with Krebs buffer only, neither HO-1 nor HO-2 expression was afftected in preparations perfused for 30 min with buffer containing ANG II or PMA ( Fig. 3 ). On the other hand, as shown in Fig. 4, the expression of HO-1 protein increased ( P
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+ n9 ?/ s/ u5 m: j# S( {Fig. 3. Assessment of heme oxygenase (HO)-1 and -2 expression by immunoblotting of proteins in rat kidneys perfused during a 30-min experimental period with oxygenated Krebs bicarbonate buffer only ( n = 4), buffer containing ANG II (1 µmol/l; n = 4), or buffer containing PMA (0.2 µmol/l; n = 4). Densitometry data (%control) are means ± SE.$ X1 x# N) L6 ]
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Fig. 4. Assessment of HO-1 and -2 expression by immunoblotting of proteins in rat kidneys perfused during a 120-min experimental period with oxygenated Krebs bicarbonate buffer only ( n = 4), buffer containing ANG II (1 µmol/l; n = 4), or buffer containing PMA (0.2 µmol/l; n = 4). Densitometry data (%control) are means ± SE. * P 6 X7 D: M2 K& z
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Figure 5 illustrates the effects of the HO inhibitor SnMP (30 µmol/l) on perfusion pressure and the concentration of CO in the venous effluent of kidneys perfused with Krebs buffer containing and not containing ANG II (1 µmol/l). SnMP increased ( P : G0 l; v: C. ^, x
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Fig. 5. Effect of stannous mesoporphyrin (SnMP; 30 µmol/l) on PP and the concentration of CO in the venous effluent of kidneys perfused with Krebs bicarbonate buffer containing ( n = 6) and not containing ( n = 6) ANG II (1 µmol/l). Values are means ± SE. * P
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DISCUSSION
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2 l7 _- J* |, f8 O5 w4 T' pThis study shows the occurrence of CO in the venous effluent of isolated rat kidneys perfused with Krebs bicarbonate buffer and demonstrates that the concentration of CO is increased by the inclusion of ANG II or PMA in the perfusion buffer. As the rate of perfusion was maintained constant throughout the experiments, the ANG II- and PMA-induced elevation of CO concentration in the renal venous effluent is indicative of increased release of CO from the kidney. It is unlikely that the stimulatory effect of ANG II and PMA on CO release is a consequence of events triggered by the associated elevation of perfusion pressure, because phenylephrine increased perfusion pressure without stimulating CO release. Because the HO inhibitor SnMP greatly decreased the concentration of CO in the venous effluent of kidneys perfused with buffer containing and not containing ANG II, the renal release of CO in both such settings appears to be dependent on HO-catalyzed generation of CO rather that on generation of CO via an HO-independent pathway ( 33 ). That stimulation of CO release from perfused kidneys by ANG II was found to occur at concentrations (1-0.01 µmol/l) which greatly exceed those found in blood even during conditions that activate the renin-angiotensin system, i.e., 0.0001-0.0005 µmol/l ( 25 ), detracts from the notion that circulating ANG II exerts a regulatory influence on renal CO release in physiological settings. Perhaps a more likely possibility is that in physiological settings the regulation of renal CO release is linked to the level of ANG II at discrete intrarenal fluid compartments. In this regard, reported levels of ANG II in renal interstitial and proximal tubule fluids ( 25, 26, 29 ) are much higher than in blood and well within the range of concentration required for the peptide to effect stimulation of CO release in the perfused rat kidney.
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According to our study, neither ANG II nor PMA affected the renal expression of HO-2 protein, but both agents increased the expression of HO-1. Previous reports indicate that upregulation of HO-1 can be demonstrated within a few hours after exposure to the inducing stimuli ( 20, 23 ). We found renal HO-1 to be significantly increased 120 min after the onset of renal perfusion with media containing 1 µmol/l ANG II or 0.2 µmol/l PMA. Importantly, the stimulation of CO release in kidneys treated with 1 µmol/l ANG II or 0.2 µmol/l PMA was documented within 30 min after initiation of treatment, preceding the augmentation of HO-1 protein. Hence, whereas upregulation of HO-1 expression is a most likely contributor to the enhanced release of CO noted late during treatment with ANG II or PMA, stimulation of CO release at an early time, when HO-1 is not upregulated, is likely driven by one or more alternative mechanisms including increased availability of endogenous heme and/or posttranslational modification of HO-2, resulting in the augmentation of catalytic activity ( 8, 17 ).! H) L4 N) K2 r& W$ ?; Y3 ]
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The expression of HO-1 was reported to increase in the kidney, heart, and arterial vessels of rats infused with ANG II ( 3, 13, 14 ) and in cultured endothelial and proximal tubular epithelial cells exposed to the peptide ( 11, 19 ). The expression of HO-1 in several cell types also was found to increase in response to conditions that activate PKC ( 4, 30, 31 ). Therefore, it is possible that upregulation of HO-1 expression in kidneys perfused with media containing ANG II is a consequence of PKC activation. PKC also was reported to phosphorylate HO-2, augmenting its catalytic activity ( 8 ), and to promote release of CO from microvessels via a mechanism involving increased availability of endogenous heme ( 17 ).
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PKC plays a central role in ANG II signaling via AT 1 receptors ( 10 ). The ANG II-induced release of CO was blunted in kidneys perfused with buffer containing the AT 1 -receptor antagonist losartan; it also was blunted in kidneys perfused with buffer containing the PKC inhibitors staurosporine or calphostin C. These findings imply that the stimulatory action of ANG II on CO release from the kidney is linked to activation of AT 1 receptors and involves PKC. The notion that PKC contributes to ANG II-induced CO release is in keeping with our finding that a known activator of PKC, PMA, also promotes CO release from the perfused kidney. On the other hand, phenylephrine, which is known to increase vascular PKC activity ( 16 ), was found not to promote CO release from the perfused kidney. This observation is intriguing because adrenergically induced vasoconstriction in the isolated, perfused rat kidney was documentated to rely, at least in part, on a mechanism involving PKC ( 28 ). Together, the aforementioned findings imply that phenylephrine-induced activation of PKC in the perfused kidney occurs at vascular sites that are not involved in the generation of CO released into the perfusate.# R n2 P5 a$ }0 P# P& k8 Z
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Our present study demonstrates that perfusion pressure is increased after HO inhibition with SnMP, more so in kidneys perfused with buffer containing than not containing ANG II. The study also documents that the ANG II-induced elevation of perfusion pressure is intensified in kidneys undergoing perfusion with buffer containing SnMP. As the rate of perfusion was held constant, increments of perfusion pressure denote augmentation of renal vascular resistance. The greater vasoconstrictor response to HO inhibition in kidneys perfused with ANG II-containing buffer, and to ANG II in kidneys perfused with SnMP-containing buffer, may be taken as evidence that an HO product exerts a restraining influence on ANG II-induced renal vasoconstriction. This conclusion is consistent with the results of a recent study documenting intensification of the renal vasoconstrictor effect of ANG II after HO inhibition ( 27 ). HO-derived CO is a prime candidate to serve as an inhibitory modulator of the actions of ANG II on the renal vasculature. In this regard, exogenous CO was shown to dilate preglomerular renal arterial vessels ( 32 ), primarily when nitric oxide synthesis is arrested ( 27 ). However, exogenous CO was reported to elicit constriction of isolated renal interlobular arteries in which nitric oxide synthesis was not arrested by pharmacological means ( 27 ). Another HO product(s) that may serve as an inhibitory modulator of vasoconstriction is biliverdin, which was reported to dilate small mesenteric arteries preconstricted with phenylephrine ( 22 ). However, biliverdin was shown not to elicit dilation of myogenically constricted rat renal interlobular arteries ( 27 ).
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2 O( B1 s: a3 J7 C0 w; FThe notion that an HO product provides a counterregulatory influence to the effect of ANG II on renal hemodynamics is particularly important in view of reports that severe reduction of glomerular filtration rate and intensification of proteinuria result from the administration of an HO inhibitor to rats with ANG II-induced hypertension ( 3 ). Moreover, the incidence of severe impairment of renal function after unilateral renal artery clipping is increased in HO-1 null mice relative to data in control mice expressing the HO-1 gene ( 34 ). Collectively, these observations support the concept that upregulation of HO-1 in conditions featuring intense activation of the renin-angiotensin system subserves a counterregulatory function aimed at preventing or minimizing ANG II-induced renal injury ( 3 ). Upregulation of renal HO-1 also was shown to confer protection against many different types of conditions injurious to the kidney ( 2, 6, 21, 23, 24 ).: Q2 X8 e7 s1 U$ U& n
7 ~7 ^2 T0 R) I3 S" g7 tIn summary, this study in isolated, perfused rat kidneys demonstrates that ANG II induces HO-1 and increases the release of CO into the venous effluent. The ANG II-induced release of CO is linked to activation of AT 1 receptors and involves PKC. Pharmacological inhibition of HO decreases the release of CO from the kidneys and elevates renal perfusion pressure, more so in preparations perfused with buffer containing than not containing ANG II. HO inhibition also intensifies the renal vasoconstrictor effect of ANG II. These data suggest that upregulation of renal HO-1 and CO release are protagonic events in a counterregulatory mechanism that attenuates ANG II-induced renal vasoconstriction.
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GRANTS
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9 R9 g0 w _' i, Z1 N5 ]This study was supported by National Heart, Lung, and Blood Institute Grants HL-18579 and HL-34300 to A. Nasjletti and a Grant from the American Heart Association, New York Affiliate, to P. Li." e: Z& e1 \1 L( q
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We thank Jennifer Brown and Chiara Kimmel-Preuss for secretarial assistance.4 O$ O5 c6 @9 S+ S1 U
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发表于 2009-4-22 08:09
