Na+/H+ exchange regulatory factor 1 is required for ROMK1 K+ channel expression in the surface membrane of cultured M-1 cortical collecting duct cells.

The ROMK1 K+ channel, a member of the ROMK channel family, is the major candidate for the K+ secretion pathway in the renal cortical collecting duct (CCD). ROMK1 possesses a PDZ domain-binding motif at its C-terminus that is considered a modulator of ROMK1 expression via interaction with Na+/H+ exchange regulatory factor (NHERF) 1 and NHERF2 scaffold protein. Although NHERF1 is a potential binding partner of the ROMK1 K+ channel, the interaction between NHERF1 and K+ channel activity remains unclear. Therefore, in this study, we knocked down NHERF1 in cultured M-1 cells derived from mouse CCD and investigated the surface expression and K+ channel current in these cells after exogenous transfection with EGFP-ROMK1. NHERF1 knockdown resulted in reduced surface expression of ROMK1 as indicated by a cell biotinylation assay. Using the patch-clamp technique, we further found that the number of active channels per patched membrane and the Ba2+-sensitive whole-cell K+ current were decreased in the knockdown cells, suggesting that reduced K+ current was accompanied by decreased surface expression of ROMK1 in the NHERF1 knockdown cells. Our results provide evidence that NHERF1 mediates K+ current activity through acceleration of the surface expression of ROMK1 K+ channels in M-1 cells.

Proinflammatory Cytokines and Potassium Channels in the Kidney.

Proinflammatory cytokines affect several cell functions via receptor-mediated processes. In the kidney, functions of transporters and ion channels along the nephron are also affected by some cytokines. Among these, alteration of activity of potassium ion (K(+)) channels induces changes in transepithelial transport of solutes and water in the kidney, since K(+) channels in tubule cells are indispensable for formation of membrane potential which serves as a driving force for the transepithelial transport. Altered K(+) channel activity may be involved in renal cell dysfunction during inflammation. Although little information was available regarding the effects of proinflammatory cytokines on renal K(+) channels, reports have emerged during the last decade. In human proximal tubule cells, interferon-γ showed a time-dependent biphasic effect on a 40 pS K(+) channel, that is, delayed suppression and acute stimulation, and interleukin-1ß acutely suppressed the channel activity. Transforming growth factor-ß1 activated KCa3.1 K(+) channel in immortalized human proximal tubule cells, which would be involved in the pathogenesis of renal fibrosis. This review discusses the effects of proinflammatory cytokines on renal K(+) channels and the causal relationship between the cytokine-induced changes in K(+) channel activity and renal dysfunction.

Interleukin-1ß suppresses activity of an inwardly rectifying K+ channel in human renal proximal tubule cells.

We investigated the effect of interleukin-1ß (IL-1ß) on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells (RPTECs), using the patch-clamp technique and Fura-2 Ca2+ imaging. IL-1ß (15 pg/ml) acutely reduced K+ channel activity in cell-attached patches. This effect was blocked by the IL-1 receptor antagonist (20 ng/ml), an inhibitor of phospholipase C, neomycin (300 µM), and an inhibitor of protein kinase C (PKC), GF109203X (500 nM). The Fura-2 Ca2+ imaging revealed that IL-1ß increased intracellular Ca2+ concentration even after removal of extracellular Ca2+, which was blocked by an inhibitor of inositol 1,4,5-trisphosphate receptors, 2-aminoethoxydiphenyl borate (2-APB, 1 µM). Moreover, IL-1ß suppressed channel activity in the presence of 2-APB without extracellular Ca2+. These results suggest that IL-1ß suppresses K+ channel activity in RPTECs through binding to its specific receptor and activation of the PKC pathway even though intracellular Ca2+ does not increase.

Effects of cytokines on potassium channels in renal tubular epithelia.

Renal tubular potassium (K(+)) channels play important roles in the formation of cell-negative potential, K(+) recycling, K(+) secretion, and cell volume regulation. In addition to these physiological roles, it was reported that changes in the activity of renal tubular K(+) channels were involved in exacerbation of renal cell injury during ischemia and endotoxemia. Because ischemia and endotoxemia stimulate production of cytokines in immune cells and renal tubular cells, it is possible that cytokines would affect K(+) channel activity. Although the regulatory mechanisms of renal tubular K(+) channels have extensively been studied, little information is available about the effects of cytokines on these K(+) channels. The first report was that tumor necrosis factor acutely stimulated the single channel activity of the 70 pS K(+) channel in the rat thick ascending limb through activation of tyrosine phosphatase. Recently, it was also reported that interferon-γ (IFN-γ) and interleukin-1ß (IL-1ß) modulated the activity of the 40 pS K(+) channel in cultured human proximal tubule cells. IFN-γ exhibited a delayed suppression and an acute stimulation of K(+) channel activity, whereas IL-1ß acutely suppressed the channel activity. Furthermore, these cytokines suppressed gene expression of the renal outer medullary potassium channel. The renal tubular K(+) channels are functionally coupled to the coexisting transporters. Therefore, the effects of cytokines on renal tubular transporter activity should also be taken into account, when interpreting their effects on K(+) channel activity.

Interaction between Calcineurin and Ca/Calmodulin Kinase-II in Modulating Cellular Functions.

Roles of calcineurin (CaN), a Ca(2+)/calmodulin- (CaM-) dependent protein phosphatase, and Ca(2+)/CaM-dependent protein kinase-II (CaMKII) in modulating K(+) channel activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) have been investigated in renal tubule epithelial cells. The channel current through the cell membrane was recorded with the patch-clamp technique, and [Ca(2+)](i) was monitored using fura-2 imaging. We found that a CaN-inhibitor, cyclosporin A (CyA), lowered the K(+) channel activity and elevated [Ca(2+)](i), suggesting that CyA closes K(+) channels and opens Ca(2+)-release channels of the cytosolic Ca(2+)-store. Moreover, both of these responses were blocked by KN-62, an inhibitor of CaMKII. It is suggested that the CyA-mediated response results from the activation of CaMKII. Indeed, Western blot analysis revealed that CyA increased phospho-CaMKII, an active form of CaMKII. These findings suggest that CaN-dependent dephosphorylation inhibits CaMKII-mediated phosphorylation, and the inhibition of CaN increases phospho-CaMKII, which results in the stimulation of CaMKII-dependent cellular actions.

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct.

We examined the mechanisms involved in the [Ca(2+)](i) response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH(2)O (hypotonic) evoked transient increase in [Ca(2+)](i) of principal cells of rat CCDs. The [Ca(2+)](i) increase was markedly attenuated by the removal of extracellular Ca(2+)(.) The application of a P(2) purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca(2+)](i) increase. The [Ca(2+)](i) increase in response to extracellular hypotonicity was not influenced by application of Gd(3+) and ruthenium red. On the other hand, a voltage-gated Ca(2+) channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca(2+)](i) increase in the principal cells. In order to assess Ca(2+) entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn(2+). The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn(2+), indicating that a Ca(2+)-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn(2+) quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca(2+) entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca(2+)](i) elevation of principal cells in rat CCD.

Role of calcineurin-mediated dephosphorylation in modulation of an inwardly rectifying K+ channel in human proximal tubule cells.

Activity of an inwardly rectifying K(+) channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca(2+)/calmodulin (CaM)-dependent phosphatase, in modulating K(+) channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 microM) or FK520 (5 microM), significantly suppressed channel activity. Intracellular Ca(2+) concentration ([Ca(2+)]( i )) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca(2+)]( i ), we speculated that inhibiting CaN enhances Ca(2+)-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca(2+)/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 microM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside-out patches, application of CaM (0.6 microM) and CaMKII (0.15 U/ml) to the bath at 10(-6) M Ca(2+) significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K(+) channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.

Delayed and acute effects of interferon-gamma on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells.

The activity of an inwardly rectifying K(+) channel in cultured human renal proximal tubule cells (RPTECs) is stimulated and inhibited by nitric oxide (NO) at low and high concentrations, respectively. In this study, we investigated the effects of IFN-gamma, one of the cytokines which affect the expression of inducible NO synthase (iNOS), on intracellular NO and channel activity of RPTECs, using RT-PCR, NO imaging, and the cell-attached mode of the patch-clamp technique. Prolonged incubation (24 h) of cells with IFN-gamma (20 ng/ml) enhanced iNOS mRNA expression and NO production. In these cells, a NOS inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 microM), elevated channel activity, suggesting that NO production was so high as to suppress the channel. This indicated that IFN-gamma would chronically suppress channel activity by enhancing NO production. Acute effects of IFN-gamma was also examined in control cells. Simple addition of IFN-gamma (20 ng/ml) to the bath acutely stimulated channel activity, which was abolished by inhibitors of IFN-gamma receptor-associated Janus-activated kinase [P6 (1 microM) and AG490 (10 microM)]. However, l-NAME did not block the acute effect of IFN-gamma. Indeed, IFN-gamma did not acutely affect NO production. Moreover, the acute effect was not blocked by inhibition of PKA, PKG, and phosphatidylinositol 3-kinase (PI3K). We conclude that IFN-gamma exerted a delayed suppressive effect on K(+) channel activity by enhancing iNOS expression and an acute stimulatory effect, which was independent of either NO pathways or phosphorylation processes mediated by PKA, PKG, and PI3K in RPTECs.

Involvement of endogenous nitric oxide in the regulation of K+ channel activity in cultured human proximal tubule cells.

Nitric oxide (NO) modulates the activity of an inwardly rectifying K(+) channel in cultured human proximal tubule cells. In this study, we investigated which NO synthase (NOS) isoform(s) was involved in the endogenous production of NO and hence the regulation of channel activity. The patch-clamp experiments using the cell-attached mode showed that a nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM), suppressed channel activity, whereas a NOS substrate, L-arginine (500 microM), stimulated it. A neuronal NOS (nNOS)/inducible NOS (iNOS)-selective inhibitor, 1-(alpha,alpha,alpha-trifluoro-o-tolyl)-imidazole (TRIM; 100 microM), suppressed channel activity to the same extent as L-NAME. TRIM also blocked the stimulatory effect of L-arginine. In contrast, an NO donor, sodium nitroprusside (10 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (100 microM) stimulated channel activity even in the presence of TRIM. RT-PCR revealed that iNOS mRNA alone was expressed in most of the cultures, i.e., 34 out of 40. In the other 6 cases, endothelial NOS (eNOS) and iNOS mRNA were simultaneously expressed. This finding was confirmed at the protein level by Western blotting. Indeed, in the patch-clamp experiments TRIM sometimes failed to suppress the channel activity, but the following addition of L-NAME suppressed it. However, since the suppressive effect of TRIM was usually similar to that of L-NAME, the involvement of eNOS in K(+) channel regulation would be relatively low. These results suggest that iNOS plays a pivotal role in the endogenous production of NO under the basal condition, which is involved in the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells.

Noninvasive measurement of temperature and fractional dissociation of imidazole in human lower leg muscles using 1H-nuclear magnetic resonance spectroscopy.

The temperature change of the fractional dissociation of imidazole (alpha-imidazole) in resting human lower leg muscles was measured noninvasively using (1)H-nuclear magnetic resonance spectroscopy at 3.0 and 1.5 T on five normal male volunteers aged 30.6 +/- 10.4 yr (mean +/- SD). Using (1)H-nuclear magnetic resonance spectroscopy, water, carnosine, and creatine in the muscles could be simultaneously analyzed. Carnosine contains imidazole protons. The chemical shifts of water and carnosine imidazole protons relative to creatine could be used for estimating temperatures and alpha-imidazole, respectively. Using the chemical shift, the values of temperature in gastrocnemius (Gast) and soleus muscles at ambient temperature (21-25 degrees C) were estimated to be 35.5 +/- 0.5 and 37.4 +/- 0.6 degrees C (means +/- SE), respectively (significantly different; P < 0.01). The estimated values of alpha-imidazole in these muscles were 0.620 +/- 0.007 and 0.630 +/- 0.013 (means +/- SE), respectively (not significant). Alternation of the surface temperature of the lower leg from 40 to 10 degrees C significantly changed the temperature in Gast (P < 0.0001) from 38.1 +/- 0.5 to 28.0 +/- 1.2 degrees C, and the alpha-imidazole in Gast decreased from 0.631 +/- 0.003 to 0.580 +/- 0.011 (P < 0.05). However, the values of alpha-imidazole and the temperature in soleus muscles were not significantly affected by this maneuver. These results indicate that the alpha-imidazole in Gast changed significantly with alternation in muscle temperature (r = 0.877, P < 0.00001), and its change was estimated to be 0.0058/ degrees C.

Regulation of an inwardly rectifying K+ channel by nitric oxide in cultured human proximal tubule cells.

We investigated the effects of nitric oxide (NO) on activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells, using the cell-attached mode of the patch-clamp technique. An inhibitor of NO synthases, N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM), reduced channel activity, which was restored by an NO donor, sodium nitroprusside (SNP; 10 microM) or 8-bromo-cGMP (8-BrcGMP; 100 microM). However, SNP failed to activate the channel in the presence of an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM). Similarly, the SNP effect was abolished by a protein kinase G (PKG)-specific inhibitor, KT-5823 (1 microM), but not by a protein kinase A-specific inhibitor, KT-5720 (500 nM). Another NO donor, S-nitroso-N-acetyl-D,L-penicillamine (10 microM), mimicked the SNP-induced channel activation. In contrast to the stimulatory effect of SNP at a low dose (10 microM), a higher dose of SNP (1 mM) reduced channel activity, which was not restored by 8-BrcGMP. Recordings of membrane potential with the slow whole cell configuration demonstrated that l-NAME (100 microM) and the high dose of SNP (1 mM) depolarized the cell by 10.1 +/- 2.6 and 9.2 +/- 1.0 mV, respectively, whereas the low dose of SNP (10 microM) hyperpolarized it by 7.1 +/- 0.7 mV. These results suggested that the endogenous NO would contribute to the maintenance of basal activity of this K(+) channel and hence the potential formation via a cGMP/PKG-dependent mechanism, whereas a high dose of NO impaired channel activity independent of cGMP/PKG-mediated processes.

Modulation of the Ca2+-activated large conductance K+ channel by intracellular pH in human renal proximal tubule cells.

The Ca2+-activated and voltage-sensitive large conductance K+ channel (BK channel) with a slope conductance of about 300 pS is present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs). In this study we examined the effects of cytoplasmic pH (pH(i)) on activity and gating kinetics of the BK channel by using the inside-out configuration of the patch-clamp technique. At a constant cytoplasmic Ca(2+) concentration ([Ca2+]i), membrane depolarization raised channel open probability (P(o)), and lowering pH(i) shifted the P(o)-membrane potential (V(m)) relationship to the positive voltage direction. However, the value of the gating charge was not affected by changes in pH(i), suggesting that the effects of pH(i) on P(o) were not due to an alternation of the voltage sensitivity. At constant V(m), lowering pH(i) suppressed the [Ca2+]i-dependent channel activation and shifted the P(o)-[Ca2+]i relationship in the direction of higher [Ca2+]i with a reduction of maximal P(o). Furthermore, both the mean open and mean closed times of the BK channels at pH(i) 6.3 in the presence of 10(-4) M [Ca2+](i) were shorter than those at pH(i) 7.3 in the presence of 10(-5) M [Ca2+]i, even though these two different conditions gave a similar P(o). The data indicate that cytoplasmic H+ suppresses P(o) of the BK channel in RPTECs, which involves the mechanism independent of Ca2+ activation. Our preliminary kinetic analysis also supported this notion.

Protein kinase G activates inwardly rectifying K(+) channel in cultured human proximal tubule cells.

An ATP-regulated inwardly rectifying K(+) channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K(+) channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 microM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 microM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 microM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K(+) channel and producing the stimulatory effect of ANP on channel activity.

In vitro 1H-NMR spectroscopic analysis of metabolites in fast- and slow-twitch muscles of young rats.

The lactate (LAC), creatine (CRN), taurine (TAU), anserine (ANS) and carnosine (CAR) content of the masseter muscles (MM), long extensor muscles of digits (EDL) and soleus muscles (SOL) of young rats were determined using in vitro 1H-NMR spectroscopy to assess the significance of CRN, TAU, ANS and CAR in these muscles. The muscles of Wistar rats at the ages of 6, 12 and 18 weeks were dissected after decapitation and used for the metabolite analyses. The LAC and CAR content of all muscle groups showed no age dependence. The CRN content was increased age-dependently in MM but not in EDL or SOL. The LAC and CRN content was higher in MM and EDL (fast-twitch) than in SOL (slow-twitch) (P<0.01-0.001 at 18 weeks). A significant positive correlation existed between the LAC and CRN content (P<0.00001, r=0.80), suggesting that the CRN content reflects the capacity of the anaerobic glycolysis of the individual muscles. The TAU content was higher in SOL and MM than in EDL (P<0.05) and showed an approximately 1.5-fold increase with age in all three muscle groups. The ANS content was higher in EDL than in SOL and MM (P<0.001), and showed an approximately threefold increase with age in all three muscle groups. The ANS content positively correlated with the LAC content (P<0.001, r=0.41), and the chemical shift of the imidazole proton in ANS showed a correlation with the LAC content (P<0.0001, r>0.76), indicating that ANS would buffer the pH change produced by LAC. These results suggest that 1H-NMR spectroscopy would provide an adjunct method of assessing the muscle types and their development.