Báo cáo khoa học: cGMP transport by vesicles from human and mouse erythrocytes

cGMP transport by vesicles from human and mouse erythrocytes

Tác giả: Cornelia J. F. de Wolf, Hiroaki Yamaguchi, Ingrid van der Heijden, Peter R. Wielinga, Stefanie L. Hundscheid, Nobuhito Ono, George L. Scheffer, Marcel de Haas, John D. Schuetz, Jan Wijnholds and Piet Borst

Lĩnh vực: Molecular Biology, Pathology, Pharmaceutical Sciences, Neurosciences

Nội dung tài liệu: Nghiên cứu này điều tra cơ chế vận chuyển cyclic guanosine monophosphate (cGMP) qua màng tế bào hồng cầu ở người và chuột. Các nhà khoa học đã sử dụng các phương pháp như phân tích western blot, thí nghiệm vận chuyển bằng vesicle và các chuột biến đổi gen để xác định vai trò của các protein vận chuyển thuộc họ ATP-binding cassette (ABC), cụ thể là ABCC4 và ABCG2 trong quá trình này. Kết quả cho thấy ABCC4 và ABCG2 đóng góp vào việc vận chuyển cGMP ở chuột, với ABCG2 có ái lực thấp hơn nhưng tốc độ vận chuyển lớn hơn so với ABCC4. Đối với hồng cầu người, ABCC4 được xác định là yếu tố vận chuyển chính, trong khi vai trò của ABCG2 dường như không đáng kể.

Mục lục chi tiết:

• cGMP transport by erythrocytes
• Keywords
• Correspondence
• Present address
• (Received 13 September 2006, revised 20 October 2006, accepted 13 November 2006)
• doi:10.1111/j.1742-4658.2006.05591.x
• Abbreviations
• ABC, ATP-binding cassette; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; Bcrp, murine breast cancer resistance protein; BCRP, human breast cancer resistance protein; KO, knockout; MRP, multidrug resistance-associated protein; MTX, methotrexate; PG, prostaglandin.
• FEBS Journal 274 (2007) 439-450 © 2006 The Authors Journal compilation © 2006 FEBS
• cGMP transport by erythrocytes
• C. J. F. de Wolf et al.
• Fig. 1. Levels of Abccs and Abcg2 in erythrocytes from WT and KO mice. Western blot analysis of 10 µg of protein from mouse erythrocyte vesicles. Each protein was detected as described in Experimental procedures.
• Results
• ABC transporters in mouse erythrocytes
• Fig. 2. Levels of ABCCs and ABCG2 in human erythrocytes. (A) Western blot analysis of 10 µg of protein from human erythrocyte vesicles from five healthy volunteers (lanes 1-5). Each protein was detected as described in Experimental procedures. (B) Western blot analysis of 40 µg of protein from human erythrocyte vesicles from three healthy volunteers (lanes 1-3). Lane 4: 10 µg of protein from Sf9-hABCC11 cell lysate (positive control). Lane 5: 40 µg of protein from Sf9 WT cell lysate (negative control). Only results obtained with monoclonal antibody Mall-16 are shown. ABCC11 was detected as described in Experimental procedures. h. ery ves, human erythrocyte vesicles.
• cGMP transport into membrane vesicles from mouse erythrocytes
• Inhibition of cGMP transport by MRP-specific inhibitors and substrates
• cGMP transport into erythrocyte membrane vesicles from Abcc KO mice
• Abcc4 and Abcg2 transport cGMP into mouse erythrocyte vesicles
• Fig. 4. Transport of cGMP into erythrocyte vesicles from WT and KO mice. (A) Erythrocyte membrane vesicles from WT and KO mice were incubated for 30 min at 37 °C with 1.8 µм [3H]cGMP. ATP-dependent transport of cGMP into vesicles from WT mice was set to 100%. (B) Concentration-dependent transport of cGMP, 0.5-10 mm, into vesicles from WT (□), Abcc4/- (▼), Abcg2/ (●) and Abcc4/-/Abcg2 -/- (0) mice was determined over a time span of 30 min. ATP-dependent transport was calculated by subtracting the transport in the absence of ATP from that in the presence of ATP. Each value represents the mean ± SD of duplicate measurements from at least three individual mice.
• Fig. 3. Transport of cGMP into mouse and human erythrocyte vesicles. Erythrocyte membrane vesicles from five WT mice (A) or five healthy volunteers (D) were incubated for the specified times at 37 °C with 1.8 μμ [3H]cGMP. Concentration-dependent transport of cGMP into vesicles from four WT mice (B) or five healthy volunteers (E) was determined over a time span of 30 min. ATP-dependent transport was calculated by subtracting the transport in the absence of ATP from that in the presence of ATP. Each point represents the mean ATP-dependent cGMP transport ± SD. The background in the minus ATP control is illustrated in (C) and (F). Human erythrocyte vesicles 1-5 correspond to an individual subject, and are consistent throughout the figure (D, E). Erythrocyte vesicles isolated from a single mouse were sufficient to perform a single experiment in triplicate. Therefore, mice 1-4 in (A) are not the same as mice 1-4 in (B).
• The role of ABCG2/Abcg2 in cGMP transport into human and mouse erythrocyte vesicles
• Fig. 5. Effect of pH on MTX and cGMP transport into membrane vesicles from humans and from WT and KO mice. (A) Effect of pH on MTX transport. Erythrocyte membrane vesicles from humans and WT and KO mice were incubated for 10 min at 37 °C with 1 μμ [3H]MTX at either pH 7.4 (■) or pH 5.5 (). (B) Effect of pH on cGMP transport. Erythrocyte membrane vesicles from WT and KO mice were incubated for 30 min at 37 °C with 1.8 μμ [H]cGMP at either pH 7.4 (■) or pH 5.5 (). For both panels, ATP-dependent transport was calculated by subtracting the transport in the absence of ATP from that in the presence of ATP. Substrate transport into vesicles from WT mice at pH 7.4 was set to 100%. The vesicle uptake buffer was 10 mM Tris at either pH 7.4 or pH 5.5. The final pH was verified by measurement with a pH meter. Each value represents the mean ± SD of duplicate measurements from three individuals/mice. For these experiments, erythrocyte vesicles from human individuals 1, 2 and 3 from Fig. 3 were used.
• Table 1. Effect of ABCC inhibitors and substrates on cGMP transport. Membrane vesicles from human and WT mouse erythrocytes were coincubated for 30 min at 37 °C with 1.8 µм [3H]cGMP and various established ABCC inhibitors/substrates. Each value was calculated by subtracting ATP-dependent cGMP transport in the presence of inhibitor from that in the absence of inhibitor. Each value represents the mean ± SD of duplicate measurements obtained from vesicles prepared from five individual mice or six human volunteers. Sample populations were tested for normality of distribution (Gaussian distribution). Student’s t-test, with Welch’s correction for unequal variance when necessary, was performed to compare the degree of inhibition observed for each condition for mouse and human erythrocyte vesicles. The Mann-Whitney test was performed when the sample size was too small (n = 4) for an accurate estimation of sample distribution. NS, not significant.
• 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) inhibits Abcc4-specific cGMP transport but not Abcg2-specific cGMP transport
• Fig. 6. Effect of AEBSF, aprotinin and leupeptin on cGMP transport into membrane vesicles from humans and from WT and KO mice. (A) Effect of three different protease inhibitors on cGMP transport by human erythrocyte vesicles. Erythrocyte membrane vesicles were co- incubated for 30 min at 37 °C with 1.8 µм [3H]cGMP and the indicated concentration of either AEBSF, leupeptin or aprotinin. (B) Concentra- tion-dependent effect of AEBSF on cGMP transport by human erythrocyte vesicles. Erythrocyte membrane vesicles were coincubated for 30 min at 37 °C with 1.8 µм [3H]cGMP and AEBSF in the concentration range of 0.5-10 mg AEBSF per milliliter of incubation mix. (C) Effect of preincubation of human erythrocyte vesicles with AEBSF on cGMP transport. Vesicles were preincubated at room temperature with () or without (■) 1 mg of AEBSF per milliliter of incubation mix for either 0, 30 or 60 min. The length of preincubation time is shown on the x-axis. Transport reactions were initiated by addition of 4 мм АТР. (D) Concentration-dependent effect of AEBSF on cGMP transport by WT and KO mouse erythrocyte vesicles. Erythrocyte membrane vesicles from WT (■), Abcc4/- (■), Abcg2/- (□) and Abcc4/-/Abcg2-/- (■) mice were coincubated for 30 min at 37 °C with 1.8 μμ [3H]cGMP and 0, 0.1, 0.5 or 1 mg of AEBSF per milliliter of incubation mix. ATP- dependent cGMP transport activity by vesicles from WT mice without addition of AEBSF were set to 100%; all other values are relative to this value. All panels display the ATP-dependent transport of cGMP, which was calculated by subtracting the transport in the absence of ATP from that in the presence of ATP. Each value represents the mean ± SD of duplicate measurements from three individuals/mice. respectively (Fig. 4B). The ability of Abcg2 to trans- port cGMP has not been noted before. This is sup- ported not only by the experiments with the Abcg2- erythrocyte vesicles, but also by the increased cGMP transport at pH 5.5 (Fig. 5B), which is specific for the Abcg2 fraction of cGMP transport. Increased transport of MTX and resveratrol by human ABCG2 at acidic pH was first noted by Breedveld et al. [23], but it is clear from Fig. 5A that it also applies to murine Abcg2 and to the substrate cGMP (Fig. 5B), although the pH effect on cGMP transport is less pronounced than on MTX transport. Whether trans- port of cGMP by Abcg2 has any physiologic signifi- cance is doubtful, given the very low affinity of Abcg2 for this substrate. The low rate of cGMP transport by Abcg2 at substrate concentrations below 100 µm may also explain why this Abcg2 activity has not been noted before. The ability of other ABC transporters, such as ABCC4, ABCC5 and ABCC8, to transport cyclic nucleotides is accompanied by the ability to transport nucleotide analogs. Indeed, Wang et al. [31,32] have reported that ABCG2 overexpres- sion induces low-level resistance to some antiviral nucleoside analogs, presumably through increased excretion of the corresponding nucleotide analogs, and we have recently found that Abcg2 confers high- level resistance to the nucleoside analog cladribine (unpublished results). Our results for human erythrocyte vesicles confirm and extend the conclusions of Klokouzas et al. [16] and Wu et al. [18], in that cGMP transport by these vesicles is attributable to ABCC4. We found > 95% inhibition by PGE₁ and PGE2, at present the most ABCC4-specific substrates known [22], and a complete block of cGMP transport by the protease inhibitor AEBSF, which seems to be relatively specific for ABCC4, as we have not found inhibition by this com- pound of ABCG2/Abcg2 (Fig. 6). We note in passing that the inhibition of ABCC4 by AEBSF is a compli- cation that should be kept in mind, as protease inhib- itor cocktails are often used routinely in vesicular transport experiments.
• cGMP efflux from intact human erythrocytes
• Discussion
• Experimental procedures
• Animals
• Blood sampling
• cGMP efflux from intact cells
• Preparation of membrane vesicles from mouse and human erythrocytes
• Vesicular transport assay
• Generation of ABCC11 antibodies
• Western blot analysis
• Acknowledgements
• References