Department of Pharmacology, New York Medical College, Valhalla, New York 10595
Submitted 13 October 2003 ; accepted in final form 15 January 2004
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ABSTRACT |
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mitogen-activated protein kinase; protein tyrosine kinase; protein tyrosine phosphatase; potassium recycling
The expression of IGF-I and IGF-binding proteins is regulated by factors such as growth hormone (16). In addition, one important factor for the regulation of the IGF-I level in the kidney is dietary K intake. It has been shown that K restriction increases IGF-I mRNA and protein expression (9), IGF-binding protein, and IGF-I receptor in the kidney (7) and that an increase in IGF-I level is responsible for renal hypertrophy during hypokalemia (15). Because K depletion has also been shown to impair NaCl transport in the TAL (22, 30, 33), we speculate that an increase in IGF-I level may also be involved in the inhibition of transport function in the TAL during hypokalemia. However, the role of IGF-I in the regulation of epithelial transport in the TAL is not completely understood. Therefore, the goal of the present study is to explore the effect of IGF-I on the apical K channels. There are three types of K channels: renal outer medulla K+ (ROMK)-like (30 pS), the intermediate conductance (70 pS), and the Ca2+-dependent, large-conductance K channel (5, 31, 37). However, the 30- and the 70-pS K channels are mainly responsible for K recycling across the apical membrane, whereas the Ca2+-dependent, large-conductance K channel is involved in the regulation of cell volume (31). Although the molecular nature of the 70-pS K channel is not completely understood, it is most likely that ROMK is a key component of the 70-pS K channel because no 70-pS K channel has been found in the ROMK(/) mouse (19). Thus we investigated the effect of IGF-I on the apical 70-pS K channel, which plays an important role in K recycling across the apical membrane, and to delineate the mechanism by which IGF-I regulates the 70-pS K channel.
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METHODS |
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Patch-clamp technique.
Patch pipettes were pulled with a Narishige model PP83 vertical pipette puller and had resistances of 46 M when filled with 140 mM NaCl. The channel current was amplified by an Axon 200A patch-clamp amplifier and was low-pass filtered at 1 kHz by using an eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA). The current was passed through an Axon interface (Digitada1200) to digitize the signal, collected by an IBM-compatible Pentium computer (Gateway 2000) at a rate of 4 kHz and analyzed by using the pClamp software system 6.04 (Axon Instruments, Burlingame, CA). Channel activity was defined as NPo, a product of channel open probability (Po) and channel number (N). The NPo was calculated from data samples of 60-s duration in the steady state as follows
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Solution and statistics.
The pipette solution contained (in mM) 140 KCl, 1.8 Mg2Cl, and 5 HEPES (pH = 7.4). IGF-I, calphostin C, N-nitro-L-arginine methyl ester, and phenylarsine oxide (PAO) were obtained from Sigma, whereas PD-98059 and herbimycin A were purchased from Biomol (Plymouth Meeting, PA). PAO and herbimycin A were dissolved in DMSO. The final concentration of DMSO was <0.1% and had no effect on channel activity. The data are presented as means ± SE. We used paired Student's t-test to determine the statistical significance. If P value is <0.05, the difference is considered to be significant.
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RESULTS |
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After demonstrating that IGF-I has a dual effect on the 70-pS K channel, we explored the mechanism by which IGF-I stimulates the 70-pS K channel. Stimulation of IGF-I receptor has been shown to activate protein tyrosine phosphatase (PTP) by enhancing tyrosine phosphorylation (2, 17, 23) or to increase nitric oxide (NO) release (38, 42). Our previous experiments have shown that an increase in NO release or activation of PTP stimulates the channel activity (12, 20). Therefore, we examined the effect of IGF-I on the 70-pS K channel in the presence of PAO, an inhibitor of PTP. Although the channel activity in the presence of PAO (1 µM) is lower than the control value (without PAO), inhibition of PTP did not abolish the stimulatory effect of IGF-I (Fig. 4). From inspection of Fig. 4, it is apparent that addition of 25 nM IGF-I increased NPo from 0.18 ± 0.04 to 1.27 ± 0.2 (P < 0.001, n = 6). To determine whether the stimulatory effect of IGF-I is mediated by a NO-dependent pathway, the effect of IGF-I on the 70-pS K channel has been examined in the presence of 0.5 mM N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS). Figure 5 is a typical recording showing the effect of 25 nM IGF-I on channel activity. We confirmed the previous finding that inhibition of NOS decreased the channel activity (20). However, inhibition of NOS also did not block the stimulatory effect of IGF-I because IGF-I significantly increased NPo from 0.11 ± 0.02 to 0.8 ± 0.2 (P < 0.05, n = 4).
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DISCUSSION |
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Two types of K channels, 30 and 70 pS, have been identified to be the main contributors to the apical K conductance under physiological conditions (5, 36). Moreover, it is speculated from a rough estimation that the apical 70-pS K channel could contribute as much as 70% of the apical K conductance in the TAL from rats on a high-K diet (21). Although the molecular nature of the 70-pS K channel in the TAL is not clear, it is most likely that Kir1 (ROMK) is also an essential component of the 70-pS K channel because K-channel activity is absent in the apical membrane of the TAL in the ROMK-knockout mice (19). The apical K channel plays an important role in K recycling, which is essential for the function of the Na-K-Cl cotransporter. Na and K enter the cell via the Na-K-Cl cotransporter, and Na leaves the cell via a basolateral Na-K-ATPase. Because K concentration in the luminal fluid of the TAL is one order lower than that of Na and Cl, K must be recycled through the apical K channel. The importance of K recycling in maintaining NaCl absorption is best demonstrated in ROMK knockout mice in which Na transport in the loop of Henle is severely impaired (19). Because IGF-I has an effect on the 70-pS K channel in the TAL, we speculate that IGF-I should have an important effect on Na transport in the TAL.
The effect of IGF-I on the 70-pS K channel is biphasic: low concentrations of IGF-I (<100 nM) stimulate, whereas high concentrations (>200 nM) inhibit the 70-pS K channel. Relevant to this observation is the report that, in blastocytes, low concentrations of IGF-I or insulin stimulate glucose uptake, whereas high concentrations decrease glucose uptake (6). It has been proposed that high concentrations of IGF-I-induced decrease in glucose uptake are the result of a downregulation of IGF-I receptors. However, this mechanism is not supported by the observation that inhibition of PTK can abolish the IGF-induced inhibition of channel activity. Thus it is possible that high concentrations of IGF-I may activate additional signal transduction pathways, such as the PTK-dependent pathway. For instance, our laboratory (21) has shown previously that low concentrations of angiotensin II inhibit the apical K channel in the TAL by stimulation of 20-hydroxyeicosatetraenoic acid formation, whereas high concentrations of angiotensin II lead to activation of the NO-dependent pathway and stimulation of channel activity. Also, the effect of angiotensin II on the Na/H exchanger in the rat proximal tubule has been found to be concentration dependent (18, 34, 35). Although herbimycin A is a specific PTK inhibitor (27), it is possible that herbimycin A may have an effect other than inhibition of PTK. However, the observation that the effect of herbimycin A was enhanced in K-restricted rats that have a high-PTK expression (12) suggests that the effect of herbimycin A is the result of inhibition of PTK.
IGF-I has been demonstrated to play an important role in the regulation of cell proliferation (16). In addition, IGF-I has also been suggested to regulate the function of ion transporters in a variety of tissues. IGF-I has been shown to stimulate the KCl cotransporter in skeletal muscle (40) and to increase transepithelial Na transport in the urinary bladder (3, 4). In addition, IGF-induced proliferation of HEK cells is related to activation of voltage-gated K channels (11). IGF-I has also been reported to stimulate glibenclamide-sensitive K channels in follicle-enclosed Xenopus oocytes (26). Although IGF-I has been shown to regulate the function of a variety of ion transporters, the mechanism by which IGF-I regulates the ion carriers and channels is different. The effect of IGF-I on voltage-gated K channels is mediated by stimulation of phosphatidylinositol 3-kinase (11). IGF-I has been reported to decrease K-channel activity by a p38 MAP kinase-dependent pathway in rat brain stem neurons (24). The present observation, that inhibition of ERK-dependent MAP kinase abolished the stimulatory effect of IGF-I, strongly suggests that IGF-induced activation of the 70-pS K channel is mediated by ERK/MAP kinase. ERK/MAP kinase is a family of serine/threonine protein kinases (25) and has been shown to play an important role in cell proliferation, cell differentiation, and cell death. There are two possibilities by which MAP kinase regulates the 70-pS K-channel activity. MAP kinase may directly regulate the channel activity by phosphorylation of the channel proteins or associate protein. Alternatively, MAP kinase can modulate the activity of other signaling molecules, which, in turn, regulate the channel activity. Relevant to this possibility is the report that IGF-I enhances glucose transport in retinal endothelial cells by MAP kinase and PKC and that MAP kinase is the upstream of the PKC-dependent pathway (8). Further experiments are needed to delineate the mechanism by which MAP kinase regulates the 70-pS K channel. The role of ERK/MAP kinase in mediating the effect of IGF-I on the 70-pS K channel is established by experiments in which application of PD-98059 and U0126 abolished the effect of IGF-I. Although PD-98059 and U0126 are highly selective ERK1/2/MAP kinase inhibitors and have no significant effect on p38 MAP kinase and c-Jun NH2-terminal kinase. However, we cannot exclude the possibility that MAP kinase other than ERK1/2 is also involved in mediating the stimulatory effect of IGF-I on the 70-pS K channel.
Whereas the stimulatory effect of IGF-I is mediated by MAP kinase, three lines of evidence indicate that the inhibitory effect of IGF-I on the 70-pS K channel is mediated by stimulation of PTK but not by PKC. First, inhibition of PTK abolished the IGF-induced inhibition of the 70-pS K channel. Second, IGF can still inhibit the channel activity in the presence of calphostin C. Finally, herbimycin A treatment reversed the IGF-induced inhibition.
The physiological importance of biphasic effects of IGF-I on the 70-pS K channel in the TAL is not clear. It is possible that IGF-I stimulates under control conditions, whereas it inhibits Na absorption when IGF-I concentration is elevated. It has been reported that the plasma concentrations of IGF-I are between 340 and 400 ng/ml or 45 and 50 nM under physiological conditions (9). However, it has been shown that IGF-I peptide concentration increased more than twofold in the K-restricted rats by a mechanism involving augmentation of IGF-binding proteins in the kidney (15). Thus it is possible that IGF concentration may increase to the extent such that IGF-I may suppress the transport function in the TAL during hypokalemia in which Na and Cl transport in the loop of Henle were impaired (22, 30, 33). The second role of IGF-I may be involved in activation of PTK activity induced by low-K intake (39). We have previously shown that low-K intake increases activity of Src family PTK. Because high concentrations of IGF-I stimulate PTK activity, it is possible that an increase in the IGF-I signaling pathway may be the upstream of the c-Src-dependent signal transduction pathway. Further experiments are required to explore this possibility.
In conclusion, IGF-I at low concentrations stimulates, whereas at high concentrations it inhibits, the apical 70-pS K channel in the TAL. The stimulatory effect of IGF-I is mediated by MAP kinase, whereas the inhibitory effect is due to stimulation of PTK.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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