Received Accepted This work was supported by the National Natural Science Foundation of China (No ).

Transcription

1 Acta Physiologica Sinica, August 25, 2012, 64(4): Research Paper Involvement of protein kinase A activation and phospholipase A 2 inhibition in the adenosine-activated basolateral 50 ps K + channels in the thick ascending limb of the rat kidney SUI Hong-Yu 1,*, LUAN Hai-Yan 1, LIU Yu-Jie 2 1 Department of Physiology, Jiamusi University, Jiamusi , China; 2 Department of Pharmacology, Harbin Medical University, Harbin , China Abstract: The present study was designed to investigate the role of protein kinase A (PKA) and phospholipase A 2 (PLA 2 ) in the stimulating effect of adenosine on the basolateral 50 ps K + channels in the thick ascending limb (TAL) of the rat kidney. Under the anatomic microscope, the TAL was dissected. The current of 50 ps K + channels were recorded by patch clamp technology. The protein expression of phosphorylated PKA and phosphorylated PLA 2 were examined by Western blot. The results showed that cyclohexyladenosine (CHA), an analog of adenosine, increased the 50 ps K + channel activity (P < 0.05). In the presence of H8, an antagonist of PKA, CHA did not affect the 50 ps K + channel activity. In the presence of AACOCF3 (an antagonist of PLA 2 ), CHA did not further increase the 50 ps K + channel activity. CHA increased phosphorylation level of PKA, whereas inhibited phosphorylation of PLA 2 in the TAL of the rat kidney (P < 0.01). Furthermore, after blocking the PLA 2 with AACOCF3, CHA still increased the expression of phosphorylated PKA. On the contrary, CHA did not obviously change the expression of phosphorylated PLA 2 after H8 pretreatment. The results suggest that the stimulation of basolateral 50 ps K + channels by CHA is mediated by the activation of PKA followed by the inhibition of PLA 2 in the TAL of the rat kidney. Key words: K + channel; adenosine; protein kinase A; phospholipase A 2 ; thick ascending limb 蛋白激酶 A 激活和磷脂酶 A 2 抑制参与腺苷对大鼠肾髓袢升支粗段管周膜 50 ps 钾通道的易化效应 1,* 隋洪玉, 栾海艳 1 2, 刘玉洁 1 佳木斯大学基础医学院生理教研室, 佳木斯 ; 2 哈尔滨医科大学药学院, 哈尔滨 摘要 : 本文旨在研究蛋白激酶 A (protein kinase A, PKA) 和磷脂酶 A 2 (phospholipase A 2, PLA 2 ) 在腺苷对髓袢升支粗段管周膜 50 ps 钾通道刺激过程中的作用 解剖显微镜下分离髓袢升支粗段, 膜片钳技术记录 50 ps 钾通道电流, 免疫印迹技术测定 PKA 及 PLA 2 磷酸化水平的蛋白表达情况 结果显示, 环己基腺苷 (cyclohexyladenosine, CHA, 一种腺苷类似物 ) 可增加 50 ps 钾通道的活性 (P < 0.05) 在 PKA 阻断剂 H8 的存在下,CHA 不再增加 50 ps 钾通道的活性 在 PLA 2 阻断剂 AACOCF3 存在下,CHA 不再增加 50 ps 钾通道的活性 CHA 可以增加髓袢升支粗段中 PKA 的磷酸化水平, 而抑制 PLA 2 的磷酸化水平 (P < 0.01) 在 AACOCF3 存在下,CHA 依旧可以增加 PKA 的磷酸化水平 (P < 0.01); 在 H8 存在下,CHA 不再抑制 PLA 2 的磷酸化水平 (P > 0.05) 以上结果提示, 腺苷对髓袢升支粗段管周膜 50 ps 钾通道的刺激作用是通过先激活 PKA, 而后再抑制 PLA 2 途径而实现的 关键词 : 钾通道 ; 腺苷 ; 蛋白激酶 A; 磷脂酶 A 2 ; 髓袢升支粗段中图分类号 :R Received Accepted This work was supported by the National Natural Science Foundation of China (No ). * Corresponding author. Tel: ; suihongyuhappy@163.com

2 450 Adenosine is the important local active substance in the body, and it plays an important role in regulating renal hemodynamics, tubuloglomerular feed back and renin release and also is involved in regulating renal epithelial transport in several nephron segments including the proximal tubule and thick ascending limb (TAL) [1,2]. In the TAL, adenosine can inhibit the transportation of NaCl. Basolateral K + channels play a key role in generating the cell membrane potential in the TAL [3]. Because the basolateral membrane potential determines the electrochemical gradient for Cl exit across the basolateral membrane, alteration of the cell membrane potential should affect the Cl exit across the basolateral membrane, thus indirectly affecting the activity of the Na + /Cl /K + cotransporter in the apical membrane. Several types of basolateral K + channels have been identified with patch-clamp experiments [4,5]. It has been confirmed that a 50 ps inwardly rectifying K + channel is highly active in the basolateral membrane [6]. Although the 50 ps K + channel is abundant in the basolateral membrane, the mechanism by which basolateral K + channels is regulated is not completely understood. Previous study has also shown that adenosine activates the basolateral 50 ps K + channels by a PKA-dependent pathway [6]. And the effect of adenosine on the 50 ps K + channels was mimiced by the cyclohexyladenosine (CHA) which is an analog of anenosine [6]. In addition, we found that CHA has no additional effect on the 50 ps K + channels in the presence of AACOCF 3 (PLA 2 antagonist) with patch-clamp experiments. And AA- COCF 3 can increase the activity of 50 ps K + channels [7]. Thus, the main goal of the present study is to examine the effect of CHA on the PKA and PLA 2 activation and to investigate the connection between PKAand PLA 2 -dependent pathways by which adenosine regulates the K + channels. 1 MATERIALS AND METHODS Acta Physiologica Sinica, August 25, 2012, 64(4): Animals Pathogen-free Sprague-Dawley (SD) rats of either sex, 5 6 weeks old, were purchased from the animal facility of the Second Affiliated Hospital of Harbin Medical University. The rats were kept on a normal rat chow and were free to access to water. The animal experiment was approved by the Medical Ethics Committee of Jiamusi University. 1.2 Agents and solutions CHA, H8 (an antagonist of PKA), AACOCF3, collagenase and polylysine were purchased from Sigma-Aldrich Co. (USA). The antibody of p-pka, p-pla 2, PKA and PLA 2 were purchased from Santa Cruz Biotechnology, Inc. (USA). ECL chemiluminescence kit was from Pierece Co. (USA). Prestained Marker was from Fermentas, Inc. (Lithuania). HEPES-buffered NaCl solution contained (in mmol/l): 140 NaCl, 5 KCl, 1.5 MgCl 2, 1.8 CaCl 2, and 10 HEPES (ph 7.4). Pipette solution contained (in mmol/l): 140 KCl, 1.8 MgCl 2, and 10 HEPES (ph 7.4). 1.3 Preparation of the TAL The rats were sacrificed by cervical dislocation, and the kidneys were removed for dissecting the medullary TAL. The isolated medullary TAL tubules were incubated in the HEPES-buffered NaCl solution containing type 1A collagenase (1 mg/ml) at 37 C for min. The collegenase-digested TAL was transferred onto a 5 mm 5 mm cover glass coated with polylysine to immobilize the tubule. The cover glass was placed in a chamber mounted on an inverted microscope (Nikon) and the tubules were superfused with HEPES-buffered NaCl solution (in mmol/l: 140 NaCl, 5 KCl, 1.5 MgCl 2, 1.8 CaCl 2, and 10 HEPES, ph 7.4) 1.4 Patch-clamp technique Patch-clamp electrodes were made using a Narishige (PP-81) puller with thick-wall glass capillaries (Degan, Minneapolis, MN, USA) and being filled with 140 mmol/l KCl. An Axon 200A patch-clamp amplifier was used for the patch-clamp experiments. The current was low-pass filtered at 0.5 khz and digitized by an Axon interface (Digidata 1200). Data were sampled by an IBM-compatible Pentium computer at a rate of 4 khz and analyzed by using pclamp software system 9 (Axon Instruments, Burlingame, CA, USA). Channel activity was defined as NP o, a product of channel open probability (P o ) and channel number (N). The NP o was calculated from data samples of 90-s duration in the steady state as follow: NP o = (1t 1 + 2t 2...it i ), where t i is the fractional open time spent at each of the observed current levels. 1.5 Western blot The TAL was fully lysed by RIPA lysis buffer containing PMSF and phosphatase inhibitor to get protein samples. Protein samples (50 μg) were separated by electrophoresis at 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in

3 SUI Hong-Yu et al.: Involvement of PKA and PLA 2 in Basolateral 50 ps K + Channels in TAL 451 Tween-Tris-buffered saline (TBS-T), rinsed, and washed with 0.1% Tween-TBS. The membranes were incubated with the primary antibody (1:500) at 4 C for 12 h. After washing four times (10 min for each wash) with TBS-T, the membrane was incubated with the secondary antibody (1:8 000) for additional 1 h. ECL plus was used to detect the protein bands. 1.6 Statistical analysis Data were shown as means ± SD. We used Student s t- tests to determine the significance between the control and experimental periods. P < 0.05 was considered to be significant. 2 RESULTS 2.1 Effect of CHA on the 50 ps K + channels As shown in Fig.1, the addition of CHA significantly increased the NP o of basolateral K channels from (0.17 ± 0.05) to (0.38 ± 0.09) (P < 0.05). As shown in Fig. 2, the NP o of basolateral K channels with H8 treatment alone was 0.22 ± After the addition of 10 μmol/l CHA, the NP o of basolateral K channels (0.25 ± 0.05) did not significantly increase in the presence of H8. As shown in Fig. 3, the NP o of basolateral K channels Fig. 1. Effect of CHA (10 μmol/l) on the basolateral 50 ps K + channels activity of thick ascending limb (TAL). The experiments were performed in a cell-attached patch. Top trace shows the experimental time course. Two parts of the trace, indicated by numbers, are extended to show the fast time resolution. The holding potential was 0 mv, and the channel closed current is indicated by C. Means ± SD, n = 5. * P < 0.05 vs control group. Fig. 2. Effect of CHA (10 μmol/l) on the basolateral 50 ps K + channels activity of thick ascending limb (TAL) in the presence of H8 (5 μmol/l). The experiments were performed in a cell-attached patch. Top trace shows the experimental time course. Two parts of the trace, indicated by numbers, are extended to show the fast time resolution. The holding potential was 0 mv, and the channel closed current is indicated by C. Means ± SD, n = 5.

4 452 Acta Physiologica Sinica, August 25, 2012, 64(4): Fig. 3. Effect of CHA (10 μmol/l) on the basolateral 50 ps K + channels activity of thick ascending limb (TAL) in the presence of AACOCF3 (5 μmol/l). The experiments were performed in a cell-attached patch. Top trace shows the experimental time course. Two parts of the trace, indicated by numbers, are extended to show the fast time resolution. The holding potential was 0 mv, and the channel closed current is indicated by C. Means ± SD, n = 5. Fig. 4. Effect of CHA (10 μmol/l) on the expression level of p-pka in the thick ascending limb (TAL) detected by Western blot. Means ± SD, n = 4. * P < 0.01 vs control group. Fig. 5. Effect of CHA (10 μmol/l) on the p-pla 2 in the thick ascending limb (TAL) detected by Western blot. Means ± SD, n = 4. * P < 0.01 vs control group. with AACOCF 3 treatment alone was 0.31 ± After the addition of 10 μmol/l CHA, the NP o of basolateral K channels (0.34 ± 0.07) did not significantly increase in the presence of AACOCF Effect of CHA on the p-pka expression in the TAL As compared with control group (without CHA treatment), the expression level of p-pka in the TAL increased at 5 and 15 min after CHA addition (P < 0.01), but did not change at 30 min and 1 h after CHA treatment (Fig. 4). 2.3 Effect of CHA on the p-pla 2 expression in the TAL The effect of CHA on the expression level P-PLA 2 at the different time point were shown in Fig. 3. As compared with control group, the expression level of p-pla 2 decreased at 5, 15 and 30min after CHA addition (n = 4, P < 0.01), but did not change at 1 h. 2.4 Effect of AACOCF 3 on PKA activation by CHA After 15 min of AACOCF 3 pretreatment, TAL were

5 SUI Hong-Yu et al.: Involvement of PKA and PLA 2 in Basolateral 50 ps K + Channels in TAL 453 Fig. 6. Effect of CHA (10 μmol/l) on the expression level of p-pka in the thick ascending limb (TAL) in the presence of AACOCF3 (5 μmol/l). Means ± SD, n = 4. * P < 0.01 vs control group. treated with CHA for 15 min. As shown in Fig. 6, CHA still increased the expression level of p-pka with AA- COCF 3 pretreatment (P < 0.01). 2.5 Effect of H8 on PLA 2 inhibition by CHA After 15 min of H8 pretreatment, TAL were treated with CHA for 15 min. As shown in Fig. 7, CHA didn t change the expression of P-PLA 2 with H8 pretreatment. 3 DISCUSSION The main findings of the present study are that PKA activation and PLA 2 inhibition participate in the process of adenosine-activation of basolateral 50 ps K + channels and the cross-talk of these two kinds of pathways. In cells, various signal transduction molecules interact and coordinate with each other to form signal transduction pathways. While intersection and crosstalk among different transduction pathways form signal network, which determines a variety of specific cell responses and regulates integral function of the cells. Previous study has also shown that adenosine activates the basolateral 50 ps K + channels by a PKA-dependent pathway. And the effect of adenosine on the 50 ps K + channels was mimicked by CHA which is analog of anenosine [4]. In the present study, we found that a PLA 2 -dependent pathway also participates in the stimu- Fig. 7. Effect of CHA (10 μmol/l) on the expression level of p- PLA 2 in the thick ascending limb (TAL) in the presence of H8 (5 μmol/l). Means ± SD, n = 4. lation of the basolateral 50 ps K + channels by CHA. Then, we asked how CHA exerts its effect on PKA and PLA 2 and whether cross-talk between PKA- and PLA 2 - dependent pathways exist in the mechanism. The results indicated that CHA can increase the expression level of p-pka and decrease the expression level of P- PLA 2. The agonist of PKA (dibutyl-camp, DBcAMP) [6] and the inhibitor of PLA 2 (AACOCF 3 ) [7] can increase the activity of basolateral 50 ps K + channels. This indicates that adenosine increases the activity of basolateral 50 ps K + channels by stimulating PKA-dependent pathway and inhibiting PLA 2 -dependent patheway. After blocking the PLA 2 with AACOCF 3, CHA still can increase the expression level of p-pka, whereas CHA showed no obvious effects on p-pla 2 expression level after blocking the PKA with H8. These results suggest that stimulation of basolateral 50 ps K + channels by CHA is mediated by PKA activation, which in turn inhibits PLA 2 activation in the TAL of the rat kidney. The present study provides some basis for discussing the regulating mechanism of basolateral 50 ps K + channels by adenosine. In future studies, we will study the effect of adenosine on basolateral 50 ps K + channels under high salt conditions, so as to further enrich the mechanism of the effect of adenosine on potassium channel and investigate whether blood pressure is associated with adenosine.

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