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We used patch-clamp and Western blot analysis to test whether PGF2α stimulates the basolateral 10-pS Cl− channel and thiazide-sensitive Na+-Cl− cotransporter (NCC) in the distal convoluted tubule (DCT) via a prostaglandin F receptor (FP-R). Single channel and whole cell recordings demonstrated that PGF2α stimulated the 10-pS Cl− channel in the DCT. The stimulatory effect of PGF2α on the Cl− channel was mimicked by a FP-R agonist, latanoprost, but was abrogated by blocking FP-R with AL8810. Also, the effect of PGF2α on the Cl− channel in the DCT was recapitulated by stimulating PKC but was blocked by inhibiting PKC. Furthermore, inhibition of p38 MAPK but not ERK blocked the effect of PGF2α on the 10-pS Cl− channel. Inhibition of NADPH oxidase also abrogated the stimulatory effect of PGF2α on the 10-pS Cl− channel, while the addition of 10 μM H2O2 mimicked the stimulatory effect of PGF2α on the 10-pS Cl− channel. Moreover, superoxide-related species may mediate the stimulatory effect of PGF2α on the 10-pS Cl− channel because the stimulatory effect of PGF2α and H2O2 was not additive. Western blot analysis showed that infusion of PGF2α in vivo not only increased the expression of FP-R but also increased the expression of total NCC and phosphorylated NCC. We conclude that PGF2α stimulates the basolateral 10-pS Cl− channel in the DCT by activating FP-R through PKC/p38 MAPK and NADPH oxidase-dependent pathways. The stimulatory effects of PGF2α on the Cl− channel and NCC may contribute to PGF2α-induced increases in NaCl reabsorption in the DCT.

Iron and aluminum water treatment residuals from a water supply plant were used as adsorbents for Tl(I) to treat thallium-containing Tl(I) wastewater and realize the resource utilization of water treatment residuals. The feasibility study results showed that Fe-WTR and Al-WTR reached adsorption equilibria within 120 min. The Langmuir model showed maximum adsorption capacities of Tl(I) on Fe-WTR and Al-WTR as 3.751 and 0.690 mg g−1 separately at an initial concentration of 5 mg L−1. The adsorption capacities of Fe-WTR and Al-WTR positively correlated with pH. The removal of Tl(I) using Fe-WTR exceeded Al-WTR; the adsorbed Tl(I) in Fe-WTR occurred primarily in the reduced state, while the Tl(I) adsorbed in Al-WTR was mainly in acid-extractable and reduced states. FTIR and XPS data showed that Tl(I) and Fe/Al-OH-functional groups formed stable surface complexes (Fe/Al-O-Tl) during adsorption, and there was no redox reaction. This confirmed that WTR is a highly efficient adsorbent for the stable removal of Tl(I), which provides a practical foundation for industrial application in Tl(I)-containing wastewater treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fibrotic interstitial lung disease with lesions confined to the lungs and is prevalent in the middle-aged and elderly population. The average survival time after diagnosis of IPF is only 3-5 years, and the mortality rate is higher than that of most tumours. IPF is called a "tumour-like disease". Entrectinib is a new oral formulation developed by Roche and was approved by the FDA to treat a wide variety of tumours. In this study, we explored the potential effects and mechanisms of entrectinib on pulmonary fibrosis in vitro and in vivo. In vivo studies showed that entrectinib is effective in alleviating bleomycin-induced pulmonary fibrosis. In vitro studies demonstrated that entrectinib dose-dependently inhibits TGF-β1/non-Smad signaling and attenuates TGF-β1-induced fibroblast activation and epithelial-mesenchymal transition (EMT). In conclusion, entrectinib blocks TGF-β1-induced lung fibroblast activation and EMT and then attenuates bleomycin-induced pulmonary fibrosis in mice.

Background: Breast cancer and gastric cancer are the two major cancers in terms of incidence among all malignancies in China. Trastuzumab is a high-priced antitumor targeted drug indicated for the treatment of human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC), metastatic breast cancer (MBC) and metastatic gastric cancer (MGC). Trastuzumab, as a price negotiation, was added to the National Reimbursement Medicine List (NRML) in China in 2017 and was the drug with the highest financial consumption among all cancer drugs in 2018. A trastuzumab biosimilar gained market authorization in China in August 2020. This study aimed to estimate the budgetary impact of switching from a trastuzumab originator to a trastuzumab biosimilar from the Chinese payer’s perspective and the number of additional patients who could be treated with the biosimilar as resulting savings in China.Methods: Five-year budgetary savings and the number of patients potentially affected were measured based on epidemiological data of breast and gastric cancers in the Chinese population available in the literature, trastuzumab market projected data in China, and original and biosimilar drug doses and costs. The robustness of the model was tested with extensive sensitivity analyses.Results: The projected budgetary savings ranged from 0.59-1.03 billion RMB over a 5-year time period, and the total cost of savings was 3.97 billion RMB. The number of additional patients who could be treated with the biosimilar according to the budget savings would be 9005-15762. Most cost savings refer to the treatment of HER2-positive EBC (86%). Sensitivity analyses showed that the prices of the trastuzumab originator and biosimilar and the number of patients treated with trastuzumab were major influencing factors.Conclusions: The introduction and penetration of a trastuzumab biosimilar in China could lead to significant budget savings. Further studies could focus on improving the refined management of drug prescriptions, particularly for HER2-positive EBC, and therefore improve the efficiency of drug costs.

We used patch-clamp and Western blot analysis to test whether PGF

The inhibition of Type II angiotensin II receptor (AT2R) or BK2R (bradykinin type II receptor) stimulates basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT) and activates thiazide-sensitive NCC (Na-Cl cotransporter). The aim of the present study is to examine the role of AT2R and BK2R in mediating the effect of HK (high dietary K + ) intake on the basolateral K + channels, NCC, and renal K + excretion. Feeding mice (male and female) with HK diet for overnight significantly decreased the basolateral K + conductance, depolarized the DCT membrane, diminished the expression of pNCC (phosphorylated NCC) and tNCC (total NCC), and decreased thiazide-sensitive natriuresis. Overnight HK intake also increased the expression of cleaved ENaC-α and -γ subunits but had no effect on NKCC2 expression. Pretreatment of the mice (male and female) with PD123319 and HOE140 stimulated the expression of tNCC and pNCC, augmented hydrochlorothiazide-induced natriuresis, and increased the negativity of the DCT membrane. The deletion of Kir4.1 not only decreased the NCC activity but also abolished the stimulatory effect of PD123319 and HOE140 perfusion on NCC activity. Moreover, the effect of overnight HK loading on Kir4.1/Kir5.1 in the DCT and NCC expression/activity was compromised in the mice treated with AT2R/BK2R antagonists. Renal clearance study showed that inhibition of AT2R and BK2R impairs renal K + excretion in response to overnight HK loading, and the mice pretreated with PD123319 and HOE140 were hyperkalemic during HK intake. We conclude that synergistic activation of AT2R and BK2R is required for the effect of overnight HK diet on Kir4.1/Kir5.1 in the DCT and NCC activity.

Stimulation of BK2R (bradykinin [BK] B2 receptor) has been shown to increase renal Na + excretion. The aim of the present study is to explore the role of BK2R in regulating Kir4.1 and NCC (NaCl cotransporter) in the distal convoluted tubule (DCT). Immunohistochemical studies demonstrated that BK2R was highly expressed in both apical and lateral membrane of Kir4.1-positive tubules, such as DCT. Patch-clamp experiments demonstrated that BK inhibited the basolateral 40-pS K + channel (a Kir4.1/5.1 heterotetramer) in the DCT, and this effect was blocked by BK2R antagonist but not by BK1R (BK B1 receptor) antagonist. Whole-cell recordings also demonstrated that BK decreased the basolateral K + conductance of the DCT and depolarized the membrane. Renal clearance experiments showed that BK increased urinary Na + and K + excretion. However, the BK-induced natriuretic effect was completely abolished in KS-Kir4.1 KO (kidney-specific conditional Kir4.1 knockout) mice, suggesting that Kir4.1 activity is required for BK-induced natriuresis. The continuous infusion of BK with osmotic pump for 3 days decreased the basolateral K + conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide-induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK inhibited NCC function. Consequently, mice receiving BK infusion for 3 days were hypokalemic. We conclude that stimulation of BK2R inhibits NCC activity, increases urinary K + excretion, and causes mice hypokalemia and that Kir4.1 is required for BK2R-mediated stimulation of urinary Na + and K + excretion.

AT2R (AngII [angiotensin II] type 2 receptor) is expressed in the distal nephrons. The aim of the present study is to examine whether AT2R regulates NCC (Na-Cl cotransporter) and Kir4.1 of the distal convoluted tubule. AngII inhibited the basolateral 40 pS K channel (a Kir4.1/5.1 heterotetramer) in the distal convoluted tubule treated with losartan but not with PD123319. AT2R agonist also inhibits the K channel, indicating that AT2R was involved in tonic regulation of Kir4.1. The infusion of PD123319 stimulated the expression of tNCC (total NCC) and pNCC (phosphorylated NCC; Thr 53 ) by a time-dependent way with the peak at 4 days. PD123319 treatment (4 days) stimulated the basolateral 40 pS K channel activity, augmented the basolateral K conductance, and increased the negativity of distal convoluted tubule membrane. The stimulation of Kir4.1 was essential for PD123319-induced increase in NCC because inhibiting AT2R increased the expression of tNCC and pNCC only in wild-type but not in the kidney-specific Kir4.1 knockout mice. Renal clearance study showed that thiazide-induced natriuretic effect was larger in PD123319-treated mice for 4 days than untreated mice. However, this effect was absent in kidney-specific Kir4.1 knockout mice which were also Na wasting under basal conditions. Finally, application of AT2R antagonist decreased the renal ability of K excretion and caused hyperkalemia in wild-type but not in kidney-specific Kir4.1 knockout mice. We conclude that AT2R-dependent regulation of NCC requires Kir4.1 in the distal convoluted tubule and that AT2R plays a role in stimulating K excretion by inhibiting Kir4.1 and NCC.

The stimulation of β–adrenergic receptor increases thiazide-sensitive NaCl cotransporter (NCC), an effect contributing to salt-sensitive hypertension by sympathetic stimulation. We now test whether the stimulation of β–adrenergic receptor-induced activation of NCC is achieved through activating basolateral Kir4.1 in the distal convoluted tubule (DCT). Application of norepinephrine increased the basolateral 40 pS K + channel (Kir4.1/Kir5.1 heterotetramer) in the DCT. The stimulatory effect of norepinephrine on the K + channel was mimicked by cAMP analogue but abolished by inhibiting PKA (protein kinase A). Also, the effect of norepinephrine on the K + channel in the DCT was recapitulated by isoproterenol but not by α–adrenergic agonist and blocked by propranolol, suggesting that norepinephrine effect on the K + channel was mediated by β–adrenergic receptor. The whole-cell recording shows that norepinephrine and isoproterenol increased DCT K + currents and shifted the K + current ( I K ) reversal potential to negative range (hyperpolarization). Continuous norepinephrine perfusion (7 days) increased DCT K + currents, hyperpolarized I K reversal potential, and increased the expression of total NCC/phosphorylated NCC, but it had no significant effect on the expression of NKCC2 (type 2 Na-Cl-K cotransporter) and ENaC-α (epithelial Na channel-α subunit). Renal clearance study demonstrated that norepinephrine perfusion augmented thiazide-induced urinary Na + excretion only in wild-type but not in kidney-specific Kir4.1 knockout mice, suggesting that Kir4.1 is required for mediating the effect of norepinephrine on NCC. However, norepinephrine perfusion did not affect urinary K + excretion. We conclude that the stimulation of β–adrenergic receptor activates the basolateral Kir4.1 in the DCT and that the activation of Kir4.1 is required for norepinephrine-induced stimulation of NCC.

Stimulation of bradykinin (BK) B2 receptor (BK2R) has been shown to increase renal Na(+) excretion. The aim of the present study is to explore the role of BK2R in regulating Kir4.1 and Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Immunohistochemical studies demonstrated that BK2R was highly expressed in both apical and lateral membrane of Kir4.1 positive tubules such as DCT. Patch-clamp experiments demonstrated that BK inhibited the basolateral 40 pS K(+) channel (a Kir4.1/5.1 heterotetramer) in the DCT and this effect was blocked by BK2R antagonist but not by BK1R antagonist. Whole-cell recordings also demonstrated that BK decreased the basolateral K(+) conductance of the DCT and depolarized the membrane. Renal clearance experiments showed that BK increased urinary Na(+) and K(+) excretion. However, the BK-induced natriuretic effect was completely abolished in kidney-specific conditional Kir4.1 knockout mice, suggesting that Kir4.1 activity is required for BK-induced natriuresis. The continuous infusion of BK with osmotic pump for 3 days decreased the basolateral K(+) conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide-induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK inhibited NCC function. Consequently, mice receiving BK infusion for 3 days were hypokalemic. We conclude that stimulation of BK2R inhibits NCC activity, increases urinary K(+) excretion and causes mice hypokalemic and that Kir4.1 is required for BK2R-mediated stimulation of urinary Na(+) and K(+) excretion.

We used the patch-clamp technique to examine the effect of angiotensin II (ANG II) on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. Application of ANG II increased the channel activity and the current amplitude of the basolateral 50-pS K channel. The stimulatory effect of ANG II on the K channels was completely abolished by losartan, an inhibitor of type 1 angiotensin receptor (AT1R), but not by PD123319, an AT2R antagonist. Moreover, inhibition of phospholipase C (PLC) and protein kinase C (PKC) also abrogated the stimulatory effect of ANG II on the basolateral K channels in the TAL. This suggests that the stimulatory effect of ANG II on the K channels was induced by activating PLC and PKC pathways. Western blotting demonstrated that ANG II increased the phosphorylation of c-Src at tyrosine residue 416, an indication of c-Src activation. This effect was mimicked by PKC stimulator but abolished by calphostin C. Moreover, inhibition of NADPH oxidase (NOX) also blocked the effect of ANG II on c-Src tyrosine phosphorylation. The role of Src-family protein tyrosine kinase (SFK) in mediating the effect of ANG II on the basolateral K channel was further suggested by the experiments in which inhibition of SFK abrogated the stimulatory effect of ANG II on the basolateral 50-pS K channel. We conclude that ANG II increases basolateral 50-pS K channel activity via AT1R and that activation of AT1R stimulates SFK by a PLC-PKC-NOX-dependent mechanism.

Kcnj10 encodes the inwardly rectifying K+channel 4.1 (Kir4.1) and is expressed in the basolateral membrane of late thick ascending limb, distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). In the present study, we perform experiments in postneonatal day 9 Kcnj10−/−or wild-type mice to examine the role of Kir.4.1 in contributing to the basolateral K+conductance in the CNT and CCD, and to investigate whether the disruption of Kir4.1 upregulates the expression of the epithelial Na+channel (ENaC). Immunostaining shows that Kir4.1 is expressed in the basolateral membrane of CNT and CCD. Patch-clamp studies detect three types of K+channels (23, 40, and 60 pS) in the basolateral membrane of late CNT and initial CCD in wild-type (WT) mice. However, only 23- and 60-pS K+channels but not the 40-pS K+channel were detected in Kcnj10−/−mice, suggesting that Kir.4.1 is a key component of the 40-pS K+channel in the CNT/CCD. Moreover, the depletion of Kir.4.1 did not increase the probability of finding the 23- and 60-pS K+channel in the CNT/CCD. We next used the perforated whole cell recording to measure the K+reversal voltage in the CNT/CCD as an index of cell membrane potential. Under control conditions, the K+reversal potential was −69 mV in WT mice and −61 mV in Kcnj10−/−mice, suggesting that Kir4.1 partially participates in generating membrane potential in the CNT/CCD. Western blotting and immunostaining showed that the expression of ENaCβ and ENaCγ subunits from a renal medulla section of Kcnj10−/−mice was significantly increased compared with that of WT mice. Also, the disruption of Kir4.1 increased aquaporin 2 expression. We conclude that Kir4.1 is expressed in the CNT and CCD and partially participates in generating the cell membrane potential. Also, increased ENaC expression in medullary CD of Kcnj10−/−mice is a compensatory action in response to the impaired Na+transport in the DCT.

Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because a 10 pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5 micromol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-omega-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10 pS channel. However, inhibition of CYP-omega-hydroxylation completely abolished the effect of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-omega-hydroxylation-dependent metabolites. We conclude that arachidonic acid inhibits the 10 pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-omega-hydroxylation-dependent metabolite 20-HETE.

To study the inhibition of the inwardly rectifying basolateral 50 pS potassium channels by PGE(2) we performed patch-clamp studies on the basolateral membrane of the rat kidney thick ascending limb. PGE(2)'s effect was mimicked by the selective EP1- and EP3-receptor agonist, sulprostone, but was prevented by inhibiting protein kinase-C with calphostin-C. The mitogen-activated protein kinase inhibitor PD98059 (ERK) or SB203580 (p38) increased basal channel activity; however, while neither alone prevented the inhibitory effect of PGE(2), but using both of them together completely abolished PGE(2)'s effect on channel activity. Treatment with PGE(2) stimulated phosphorylation of both p38 and ERK in primary cultures of medullary thick ascending limb cells. The PGE(2)-mediated mitogen-activated protein kinase activation was not affected by indomethacin, but was completely blocked by calphostin-C. These studies show that inhibition of basolateral 50 pS potassium channels by PGE(2) is mediated by protein kinase-C, which in turn stimulates mitogen-activated protein kinases in the thick ascending limb of the rat kidney.

Adenosine is a molecule produced by several organs within the body, including the kidneys, where it acts as an autoregulatory factor. It mediates ion transport in several nephron segments, including the proximal tubule and the thick ascending limb (TAL). Ion transport is dictated in part by anionic chloride channels, which regulate crucial kidney functions, including the reabsorption of Na+ and Cl‑, urine concentration, and establishing and maintaining the corticomedullary osmotic gradient. The present study investigated the effects of adenosine on the mRNA expression of chloride voltage‑gated channel Kb (CLCNKB), a candidate gene involved in hypertension, which encodes for the ClC‑Kb channel. Medullary thick ascending limb (mTAL) tubules were isolated from the rat kidney, and primary cultures of mTAL cells from the mTAL tubules were established. The cells were treated with adenosine and the mRNA expression of CLCNKB was detected by reverse transcription‑quantitative polymerase chain reaction. The cells were also treated with pathways inhibitors (H8 and AACOCF3), and the protein expression of cyclic adenosine 3',5'‑monophosphate (cAMP)‑protein kinase A (PKA) and phospholipase A2 (PLA2) by were analyzed by western blotting. The findings indicated that adenosine increased the mRNA expression of CLCNKB in primary cultures of medullary TAL cells, and this stimulatory effect was regulated by the cAMP‑PKA and PLA2‑arachidonic acid (AA) pathways. The present study showed that adenosine affected the mRNA expression of CLCNKB, initially through the cAMP‑PKA pathway and then the PLA2‑AA pathway.

The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelman's syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na(+)-activated 80-150pS K(+) channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10(-/-) mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K(+) conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10(-/-) mice. Application of AVP significantly stimulated the 80-150pS K(+) channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24h significantly increased the probability of finding the 80-150pS K(+) channel and the K(+) channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K(+) channel, suggesting that cAMP-PKA pathway stimulates the 80-150pS K(+) channels. The role of the basolateral K(+) conductance in maintaining transcellular Cl(-) transport is further suggested by the finding that the inhibition of basolateral K(+) channels significantly diminished the AVP-induced stimulation of the basolateral 10pS Cl(-) channels. We conclude that vasopressin stimulates the 80-150pS K(+) channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K(+) channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K(+) conductance which is essential for the transport function in the TAL.

We used the patch-clamp technique to study the effect of insulin-like growth factor I (IGF-I) on the apical 70-pS K channel in the isolated thick ascending limb (TAL) of the rat kidney. The isolated TAL was cut open to gain access to the apical membrane. Addition of 25 nM IGF-I stimulates the apical 70-pS K channel and increases channel activity, defined by the product of channel open probability and channel number, from 0.31 to 1.21. The stimulatory effect of IGF-I is not mediated by nitric oxide- or protein tyrosine phosphatase-dependent mechanisms, because inhibition of nitric oxide synthase or blocking protein tyrosine phosphatase did not abolish the stimulatory effect of IGF-I on the 70-pS K channel. In contrast, inhibition of mitogen-activated protein (MAP) kinase with PD-98059 or U0126 abolished the stimulatory effect of IGF-I. This suggests that MAP kinase is responsible for mediating the effect of IGF-I on the apical K channels. Moreover, the effect of IGF-I on the apical 70-pS K channel is biphasic because high concentrations (>200 nM) inhibit apical 70-pS K channels. Application of 400 nM IGF-I decreased channel activity from 1.45 to 0.2. The inhibitory effect of IGF-I is not blocked by calphostin C (an inhibitor of PKC), but inhibition of protein tyrosine kinase with herbimycin A abolished the IGF-induced inhibition. We conclude that IGF-I has a dual effect on the apical 70-pS K channel in the TAL: low concentrations of IGF-I stimulate, whereas high concentrations inhibit the channel activity. The stimulatory effect of IGF-I is mediated by a MAP kinase-dependent pathway, whereas the inhibitory effect is the result of stimulation of protein tyrosine kinase.

We have shown previously that raising extracellular Ca(2)+ inhibited the apical 70-pS K channel in the thick ascending limb (TAL; Wang, W.H., M. Lu, and S.C. Hebert. 1996. Am. J. Physiol. 270:C103-C111). We now used the patch-clamp technique to study the effect of increasing the extracellular Ca(2)+ on the 70-pS K channel in the mTAL from rats on a different K diet. Increasing the extracellular Ca(2)+ from 10 microM to 0.5, 1, and to 1.5 mM in the mTAL from rats on a K-deficient (KD) diet inhibited the channel activity by 30, 65, and 90%, respectively. In contrast, raising the extracellular Ca(2)+ to 1.5 mM had no significant effect on channel activity in the mTAL from animals on a high K (HK) diet and further increasing the extracellular Ca(2)+ to 2.5, 3.5, and 5.5 mM decreased the channel activity by 29, 55, and 90%, respectively. Inhibition of the cytochrome P450 monooxygenase completely abolished the effect of the extracellular Ca(2)+ on channel activity in the mTAL from rats on a different K diet. In contrast, blocking cyclooxygenase did not significantly alter the responsiveness of the 70-pS K channel to the extracellular Ca(2)+. Moreover, addition of sodium nitropruside, a nitric oxide (NO) donor, not only increased the channel activity, but also blunted the inhibitory effect of the extracellular Ca(2)+ on the 70-pS K channel and decreased 20-hydroxyeicosatetraenoic acid (20-HETE) concentration in the mTAL from rats on a KD diet. In contrast, inhibiting NOS with L-NAME enhanced the inhibitory effect of the extracellular Ca(2)+ on the channel activity and increased 20-HETE concentration in the mTAL from rats on a high K diet. Western blot has further shown that the expression of inducible NO synthase (iNOS) is significantly higher in the renal medulla from rats on an HK diet than that on a KD diet. Also, addition of S-nitroso-N-acetylpenicillamine abolished the inhibitory effect of arachidonic acid on channel activity in the mTAL, whereas it did not block the inhibitory effect of 20-HETE. We conclude that a low dietary K intake increases the sensitivity of the 70-pS K channel to the extracellular Ca(2)+, and that a decrease in NOS activity is involved in enhancing the inhibitory effect of the extracellular Ca(2)+ on channel activity in the mTAL during K depletion.

We have used Western blot to examine the expression of cSrc protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the patch-clamp technique to determine the role of PTK in mediating the effect of dietary K intake on the small-conductance K (SK) channel in the cortical collecting duct (CCD). When rats were on a K-deficient (KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by 40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and 3 days decreased by 40, 60, and 75%, respectively. However, the protein level of PTP-1D was not significantly changed by dietary K intake. The addition of 1 μM herbimycin A increased NP o, a product of channel number ( N) and open probability ( P o) in the CCD from rats on a normal diet or on a KD diet. The increase in NP o was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively. Treatment of the CCD with herbimycin A from rats on a KD diet increased NP o per patch from the control value (0.7) to 1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD), respectively. In contrast, HK intake for as short as 1 day abolished the effect of herbimycin A. Furthermore, the expression of ROMK channels in the renal cortex was the same between rats on a KD diet or on a HK diet. Moreover, treatment with herbimycin A did not further increase NP o in the CCDs from rats on a HK diet. We conclude that dietary K intake plays a key role in regulating the activity of the SK channels and that PTK is involved in mediating the effect of the K intake on channel activity in the CCD.

Chloride channels in the basolateral membrane play a key role in Cl absorption in the thick ascending limb (TAL). The patch-clamp experiments were performed to test whether angiotensin II (AngII) increases Cl absorption in the TAL by stimulating the basolateral 10-pS Cl channels. AngII (1–100 nmol/L) stimulated the 10-pS Cl channel in the TAL, an effect that was blocked by losartan (angiotension AT1 receptor [AT 1 R] antagonist) but not by PD123319 (angiotension AT2 receptor [AT 2 R] antagonist). Inhibition of phospholipase C or protein kinase C also abolished the stimulatory effect of AngII on Cl channels. Moreover, stimulation of protein kinase C with phorbol-12-myristate-13-acetate mimicked the effect of AngII and increased Cl channel activity. However, the stimulatory effect of AngII on Cl channels was absent in the TAL pretreated with diphenyleneiodonium sulfate, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Moreover, treatment of the TAL with diphenyleneiodonium sulfate also blocked the effect of phorbol-12-myristate-13-acetate on the 10-pS Cl channel. Western blotting demonstrated that incubation of isolated TAL with AngII increased phosphorylation of p47 phox at Ser 304 , suggesting that AngII stimulates the basolateral Cl channels by increasing NADPH oxidase–dependent superoxide generation. This notion was also supported by the observation that H 2 O 2 significantly increased 10-pS Cl channel activity in the TAL. We conclude that stimulation of AT 1 R increased the basolateral Cl channels by activating the protein kinase C–dependent NADPH oxidase pathway. The stimulatory effect of AngII on the basolateral Cl channel may contribute to AngII-induced increases in NaCl reabsorption in the TAL and AngII-infuse–induced hypertension.

The aim of the present study is to test the hypothesis that insulin-like-growth factor-1 (IGF-1) plays a role in the regulation of basolateral Cl channels in the thick ascending limb (TAL). The patch-clamp experiments demonstrated that application of IGF-I or insulin inhibited the basolateral 10-pS Cl channels. However, the concentration of insulin required for the inhibition of the Cl channels by 50% (K1/2) was ten times higher than those of IGF-1. The inhibitory effect of IGF-I on the 10-pS Cl channels was blocked by suppressing protein tyrosine kinase or by blocking phosphoinositide 3-kinase (PI3K). In contrast, inhibition of phospholipase C (PLC) failed to abolish the inhibitory effect of IGF-1 on the Cl channels in the TAL. Western blot analysis demonstrated that IGF-1 significantly increased the phosphorylation of phospholipid-dependent kinase (PDK) at serine residue 241 (Ser241) and AKT at Ser473 in the isolated medullary TAL. Moreover, inhibition of PI3K with LY294002 abolished the effect of IGF-1 on the phosphorylation of PDK and AKT. The notion that the effect of IGF-1 on the 10-pS Cl channels was induced by stimulation of PDK–AKT–mTOR pathway was further suggested by the finding that rapamycin completely abolished the effect of IGF-1 on the 10-pS Cl channels in the TAL. We conclude that IGF-1 inhibits the basolateral Cl channels by activating PI3K–AKT–mTOR pathways. The inhibitory effect of IGF-1 on the Cl channels may play a role in ameliorating the ischemia-induced renal injury through IGF-1 administration.

The basolateral 50-pS K channels are stimulated by a cAMP-dependent pathway and inhibited by cytochrome P-450-omega-hydroxylase-dependent metabolism of arachidonic acid (AA) in the rat thick ascending limb (TAL). We now used the patch-clamp technique to examine whether stimulation of adenosine A2a receptor modulates the inhibitory effect of AA on the basolateral 50-pS K channels in the medullary TAL. Stimulation of adenosine A2a receptor with CGS-21680 or inhibition of phospholipase A2 (PLA2) with AACOCF3 increased the 50-pS K channel activity in the TAL. Western blot demonstrated that application of CGS-21680 decreased the phosphorylation of PLA2 at serine residue 505, an indication of inhibiting PLA2 activity. In the presence of CGS-21680, inhibition of PLA2 had no further effect on the basolateral 50-pS K channels. The possibility that CGS-21680-induced stimulation of the basolateral 50-pS K channels was partially achieved by inhibition of PLA2 in the TAL was also supported by the observation that CGS-21680 had no additional effect in the presence of AACOCF3. Moreover, stimulation of adenosine A2a receptor with CGS-21680 also abolished the inhibitory effect of AA and 20-hydroxyeicosatetraenoic acid (20-HETE) on the 50-pS K channels. The effect of CGS-21680 on AA and 20-HETE-mediated inhibition of the 50-pS K channels was mediated by cAMP because application of membrane-permeable cAMP analog, dibutyryl-cAMP, not only increased the 50-pS K channel activity but also abolished the inhibitory effect of AA and 20-HETE. We conclude that stimulation of adenosine A2a receptor increased the 50-pS K channel activity in the TAL, an effect that is achieved by suppression of PLA2 activity and 20-HETE-induced inhibition.

ST-segment elevation is the major clinical criterion for committing patients with chest pain to have emergent coronary revascularizations; however, the mechanism responsible for ST-segment elevation is unknown. In a guinea pig model of ST-segment elevation acute myocardial infarction (AMI), local application of hirudin, a thrombin antagonist, significantly decreased AMI-induced ST-segment elevation in a dose-dependent manner. Hirudin-induced (5 antithrombin units [ATU]) decrease in ST elevation was reversed by 250 nmol/L thrombin receptor activator peptide (TRAP). TRAP (250 nmol/L [100 microL]) significantly induced ST-segment elevation in hearts without AMI. The TRAP effect was blocked by 4 mg/kg glibenclamide and 4 mg/kg HMR1098 and partially blocked by 3 mg/kg 5HD. Pinacidil (0.45 mg/kg) simulated the effect of TRAP (250 nmol/L [100 microL]) on hearts without AMI. Moreover, single-channel recordings showed that TRAP induced ATP-sensitive K+ channel (KATP channel) activity, and this effect was blocked by HMR1098 but not 5HD. Finally, TRAP significantly shortened the monophasic action potential (MAP) at 90% repolarization (MAP90) and epicardial MAP (EpiMAP) duration. These effects of TRAP were completely reversed by HMR1098 and partially reversed by 5HD. Thrombin and its receptor activation enhanced ST-segment elevation in an AMI model by activating the sarcolemmal KATP channel.

Concentration of extracellular calcium ([Ca(2+)](o)) in the central nervous system decreases substantially in different conditions. It results in facilitating neuronal excitability. The goal of this study is to examine the mechanisms of enhanced neuronal excitation in low [Ca(2+)](o) in order to provide new clues to treat the hyperexcitability diseases in clinic.Whole-cell patch-clamp technique and neuron culture were used in the study.The firing threshold of cultured hippocampal neurons decreased markedly in low [Ca(2+)](o) saline. Unexpectedly, apamine and isoprenaline, antagonists of medium afterhyperpolarization (mAHP) and slow AHP (sAHP) respectively, had no statistic significant effect on excitability of neurons. TTX at a low concentration was sufficient to inhibit I(NaP), which blocked the increase of firing frequency in low [Ca(2+)](o). It also reduced the number of spikes in normal [Ca(2+)](o).These results suggest that in cultured hippocampal neurons, modulation of spiking threshold but not AHP may cause the increased excitability in low [Ca(2+)](o).

We used the patch-clamp technique to examine the effect of adenosine on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. A 50-pS inwardly rectifying K channel was detected in the basolateral membrane, and the channel activity was decreased by hyperpolarization. Application of adenosine (10 μM) increased the activity of basolateral 50 pS K channels, defined by NPo, from 0.21 to 0.41. The effect of adenosine on the 50 pS K channels was mimicked by cyclohexyladenosine (CHA), which increased channel activity by a dose-dependent manner. However, inhibition of the A1 adenosine receptor with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) failed to block the effect of CHA. In contrast, application of 8-(3-chlorostyryl) caffeine (CSC), an A2 adenosine antagonist, abolished the stimulatory effect of CHA. The possibility that the effect of adenosine and adenosine analog on the basolateral 50 pS K channel was the result of activation of the A2 adenosine receptor was also suggested by the observation that application of CGS-21680, a selected A2A adenosine receptor agonist, increased the channel activity. Also, inhibition of PKA with N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide-2HC1 abolished the stimulatory effect of CHA on the basolateral 50 pS K channel. Moreover, addition of the membrane-permeable cAMP analog increases the activity of 50 pS K channels. We conclude that adenosine activates the 50 pS K channel in the basolateral membrane of the TAL and the stimulatory effect is mainly mediated by a PKA-dependent pathway via the A2 adenosine receptor in the TAL.

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( Po) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NPo, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165–F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP o, a product of channel number ( N) and open probability ( P o), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP o of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP o of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.

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.

P95 We have used the patch clamp technique to study the effect of dietary-K intake on the apical K channels in the medullary thick ascending limb (mTAL) of rat kidneys. The channel activity, defined by NPo, of the 30 pS and 70 pS K channel was 0.18 and 0.11 in the mTAL from rats on a K-deficient diet, respectively. In contrast, NPo of the 30 pS and the 70 pS K channels increased to 0.60 and 0.80 in the tubules from animals on a high-K diet, respectively. We have also used GC/MC to measure the intracellular production of 20-hydroxyeicosanotetraenoic acid (20-HETE) in the mTAL. The concentration of 20-HETE was 0.8 pg/μg protein in the mTAL from rats on a high-K diet and increased significantly to 4.6 pg/μg protein in the tubules from rats on a K-deficient diet. Addition of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17ODYA), agents which inhibit the formation of 20-HETE, had no significant effect on the activity of the 30 pS K channels. However, DDMS/17ODYA significantly increased the activity of the apical 70 pS K channel from 0.11 to 0.91 in the mTAL from rats on a K-deficient diet. In contrast, inhibition of the cytochrome P450 metabolism of arachidonic acid (AA) increased NPo from 0.64 to 0.81 in the tubules from animals on a high-K diet. Furthermore, the concentration of 20-HETE required to block the channel activity by 50% was the same in the mTAL from rats on a high K diet as that on a K-deficient diet. This indicates that the diminished response of the 70 pS K channel to the inhibition of P450 metabolism of AA is not the result of decreasing 20-HETE sensitivity in the mTAL from rats on a high K diet. Finally, the pretreatment of the tubules with DDMS increased NPo of the 70 pS K channels in the mTAL from rats on a K-deficient diet to 0.76, a value which is not significantly different from the NPo in the tubules from rats on a high-K diet. We conclude that an increase in 20-HETE production is involved in reducing the activity of the apical 70 pS K channels in the mTAL from rats on a K-deficient diet.