Abstract
Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.
Highlights
Channels is not increased in rats infused with aldosterone or on a low sodium diet, a maneuver that increases aldosterone secretion [8]
We used Western blot analysis to detect the expression of c-Src, protein-tyrosine phosphatase (PTP)-1B, PTP-1C, and PTP-1D in the renal cortex obtained from rats on a normal chow and on a high Kϩ diet (7 days)
To explore the role of PTP in the regulation of apical small conductance K؉ (SK) channels in the collecting duct (CCD), we studied the effect of phenylarsine oxide (PAO), an inhibitor of PTP [11,12,13], on the activity of SK channels in the tubules from rats on a high Kϩ diet
Summary
Preparation of CCDs— Pathogen-free Sprague-Dawley rats of either sex (5 weeks) were used in the experiments and were purchased from Taconic Farms, Inc. (Germantown, NY). The weight of the animals used for experiments was between 100 and 120 g. Several thin slices of the kidney (Ͻ1 mm) were cut and placed on an ice-cold Ringer solution until dissection. The dissection was carried out at room temperature, and two watch-make forceps were used to isolate the single CCD. The CCDs were superfused with Hepes-buffered NaCl solution, and the temperature of the chamber was maintained at 37 Ϯ 1 °C by circulating warm water surrounding the chamber. The CCD was cut open with a sharpened micropipette to expose the apical membrane. Data stored on the tape were collected to an IBM-compatible.
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