Naomi Niisato

Regulation of an amiloride‐sensitive Na+‐permeable channel by a β2‐adrenergic agonist, cytosolic Ca2+ and Cl− in fetal rat alveolar epithelium

1 In cell‐attached patches formed on the apical membrane of fetal alveolar epithelium, terbutaline (a specific β2‐adrenergic agonist) increased the open probability (Po) of an amiloride‐sensitive Na+‐permeable non‐selective cation (NSC) channel (control, 0.03 ± 0.04; terbutaline, 0.62 ± 0.18; n= 8, P < 0.00001) by increasing the mean open time 100‐fold without any significant change in the mean closed time and without any change in the single channel conductance (control, 27.8 ± 2.3 pS; terbutaline, 28.2 ± 2.1 pS; n= 8). 2 The Po of the unstimulated channel increased when the apical membrane was depolarized due to a decrease in the closing rate and an increase in the opening rate, while the Po of the terbutaline‐stimulated channel did not depend on the membrane potential. 3 Increased cytosolic [Ca2+] also increased the Po of the channel in a manner consistent with one Ca2+‐binding site on the cytosolic surface of the channel. Terbutaline increased the sensitivity of the channel to cytosolic Ca2+ by shifting the concentration of cytosolic Ca2+ ([Ca2+]c) required for half‐maximal activation to a lower [Ca2+]c value, leading to an increase in Po. 4 An increase in the cytosolic Cl− concentration ([Cl−]c) decreased the Po of the channel consistent with two Cl−‐binding sites by increasing the closing rate without any significant change in the opening rate. Terbutaline increased Po by reducing the effect of cytosolic Cl− to promote channel closing. 5 Taken together, these observations indicate that terbutaline activates a Ca2+‐activated, Cl−‐inhibitable, amiloride‐sensitive, Na+‐permeable NSC channel in fetal rat alveolar epithelium in two ways: first, through an increase in Ca2+ sensitivity, and second, through a reduction in the effect of cytosolic Cl− to promote channel closing.

Activation of the Na+‐K+ pump by hyposmolality through tyrosine kinase‐dependent Cl− conductance in Xenopus renal epithelial A6 cells

1 We studied the regulatory mechanism of Na+ transport by hyposmolality in renal epithelial A6 cells. 2 Hyposmolality increased (1) Na+ absorption, which was detected as an amiloride‐sensitive short‐circuit current (INa), (2) Na+‐K+ pump activity, (3) basolateral Cl− conductance (Gb,Cl), and (4) phosphorylation of tyrosine, suggesting an increase in activity of protein tyrosine kinase (PTK). 3 A Cl− channel blocker, 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB), which abolished Gb,Cl, blocked the INa by inhibiting the Na+‐K+ pump without any direct effect on amiloride‐sensitive Na+ channels. Diminution of Gb,Cl by Cl− replacement with a less permeable anion, gluconate, also decreased the hyposmolality‐increased Na+‐K+ pump activity. 4 The PTK inhibitors tyrphostin A23 and genistein induced diminution of the hyposmolality‐stimulated Gb,Cl, which was associated with attenuation of the hyposmolality‐increased Na+‐K+ pump activity. 5 Taken together, these observations suggest that: (1) hyposmolality activates PTK; (2) the activated PTK increases Gb,Cl; and (3) the PTK‐increased Gb,Cl stimulates the Na+‐K+ pump. 6 This PTK‐activated Gb,Cl‐mediated signalling of hyposmolality is a novel pathway for stimulation of the Na+‐K+ pump.

Involvement of Protein Tyrosine Kinase in Osmoregulation of Na+ Transport and Membrane Capacitance in Renal A6 Cells

Abstract. Renal A6 cells have been reported in which hyposmolality stimulates Na+ transport by increasing the number of conducting amiloride-sensitive 4-pS Na+ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na+ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na+ channels. Tyrphostin A23 also abolished macroscopic Na+ currents (amiloride-sensitive short-circuit current, INa) by decreasing the elevating rate of the hyposmolality-increased INa. Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on INa by diminishing the elevating rate of the hyposmolality-increased INa, mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na+ transport by translocating the Na+ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells.

The effect of brefeldin A on terbutaline-induced sodium absorption in fetal rat distal lung epithelium

Abstract We studied the effect of brefeldin A, which inhibits the intracellular trafficking of membrane proteins from the cytosolic pool to the cell surface, on terbutaline (a β2-specific adrenergic agonist)-induced alterations in ion transport by primary monolayer cultures of fetal rat distal lung epithelium. The amiloride-sensitive short circuit current (Isc) increased 2.5-fold 50 min after application of terbutaline (10 μM) from basolateral side; this response was abolished by pretreatment with brefeldin A (1 μg/ml). Brefeldin A did not suppress the Na+/K+ pump capacity. Single channel patch clamp experiments demonstrated that terbutaline increased the density of amiloride-sensitive Na+-permeable nonselective cation channels on the apical cell membrane and this action was blocked by brefeldin A. These observations suggest that β2-specific adrenergic agonists promote the trafficking of amiloride sensitive Na+-permeable nonselective cation channels to the apical cell surface.

Regulation of Cl- transport by IBMX in renal A6 epithelium.

Abstract We studied regulation of Cl–transport by cAMP and Ca2+ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (Isc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl–channel blocker) markedly and dose-dependently inhibited the IBMX-induced Isc. Pretreatment with nifedipine (100 μM, a Ca2+ channel blocker) or 1,2-bis (o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 μM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 μM for 1 h), also reduced the IBMX-induced Isc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced Isc in cells treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced Isc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl–release is regulated by an increased cytosolic Ca2+ through nifedipine-sensitive Ca2+ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca2+ concentration but not for activation of Cl–channels, which are directly activated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl–release requires cAMP elevation in addition to an increase in the cytosolic Ca2+ concentration.

Cross talk of bumetanide-sensitive and HCO3--dependent transporters activated by IBMX in renal epithelial A6 cells.

We studied cAMP-dependent regulation of ion transport in aldosterone-untreated renal epithelial A6 cells by measuring short-circuit current (Isc). Biphasic increases in Isc, a transient phase followed by a sustained one, were elicited in response to 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase) which increased cytosolic cAMP concentration. IBMX increased the apical Cl− conductance. The sustained phase Isc induced by IBMX was reduced by 50 μm bumetanide (Na+/K+/2 Cl− cotransporter inhibitor) or 100 μm 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, an inhibitor of C1−/HCO3− exchanger). Under the normal condition, the inhibitory effect of bumetanide was much larger than that of DIDS. On the other hand, under a low Cl− condition, the effect of DIDS was more effective than that of bumetanide. Further, under a Cl−-free condition Na+/HCO3− symporter contributed to the IBMX-generated Isc. Taken together, our observations suggest that in A6 cells (i) IBMX stimulates Cl− secretion associated with an increase in apical Cl− conductances, (ii) the ionic components to generate the IBMX-induced Isc are mainly maintained by bumetanide-sensitive Na+/K+/2 Cl− cotransporter and DIDS-sensitive Cl−/HCO3− exchanger, (iii) Cl−/HCO3− exchanger coupled to Na+/HCO3− symporter under a low-Cl− condition or Na+/HCO3− symporter under a Cl−-free condition contributes to the IBMX-induced Isc, compensating for diminishment of the Na+/K+/2Cl− cotransporter-mediated Cl− secretion, (iv) IBMX increases Cl− and HCO3− conductances in the apical membrane.

Protein Phosphatase 2B-dependent Pathway of Insulin Action on Single Cl− Channel Conductance in Renal Epithelium

Abstract. The apical membrane of distal nephron epithelium (A6) has a Ca2+-dependent outwardly rectifying Cl− channel with single channel conductances of 3 pS for outward current and 1 pS for inward current under the basal condition. The single channel conductance for inward currents increased as cytosolic Ca2+ concentration ([Ca2+]c) was elevated, while the single channel conductance for outward currents did not change at the range of [Ca2+]c from 10 nm to 1 mm. Insulin (100 nm) increased the single channel conductance for the inward current by increasing the sensitivity to cytosolic Ca2+ by 400-fold, but did not affect the single channel conductance for the outward current. Further, insulin increased the open probability of the channel. These effects of insulin were completely blocked by cyclosporin-A, an inhibitor of protein phosphatase type 2B (PP2B) which dephosphorylates phospho-tyrosine in addition to phospho-serine/threonine, but not by okadaic acid, an inhibitor of protein phosphatase type 1 and 2A. Further, these effects of insulin were also completely blocked by W7, an antagonist of calmodulin which is required for activation of PP2B. Lavendustin A, an inhibitor of protein tyrosine kinase (PTK), mimicked these effects of insulin; this action of lavendustin A required 1 hr after its application, while within 30 min after its application lavendustin A had no significant effects on the single channel conductance. On the other hand, lavendustin A blocked the insulin action for a relatively short time period (i.e., within 30 min after their application). However, H89 (an inhibitor of protein kinase A) or H7 (an inhibitor of protein kinases A, C and G) did not mimic the insulin action. Application of PP2B or protein tyrosine phosphatase to the cytosolic surface of the inside-out patch membrane increased the single channel conductance and the open probability as did insulin in cell-attached patches. The insulin-induced increases in single channel conductance and open probability were reversibly decreased by application of PTK catalytic subunit in the presence of ATP through a decrease in the sensitivity to cytosolic Ca2+, but not by protein kinase A. These observations suggest that as intracellular signalling of insulin action, PP2B-mediated dephosphorylation of phospho-tyrosine of the channel protein (or channel-associated protein) is a novel mechanism for regulation of single channel conductance, and that at least two different types of PTKs regulate the channel characteristics.

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

Abstract. The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na+/K+/2Cl− cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl− cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl− cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl− cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl− cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl− cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl− cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels.

cAMP stimulates Na+ transport in rat fetal pneumocyte: involvement of a PTK- but not a PKA-dependent pathway

To study a cAMP-mediated signaling pathway in the regulation of amiloride-sensitive Na+ transport in rat fetal distal lung epithelial cells, we measured an amiloride-sensitive short-circuit current (Na+ transport). Forskolin, which increases the cytosolic cAMP concentration, stimulated the Na+ transport. Forskolin also activated cAMP-dependent protein kinase (PKA). A β-adrenergic agonist and cAMP mimicked the forskolin action. PKA inhibitors KT-5720, H-8, and myristoylated PKA-inhibitory peptide amide-(14-22) did not influence the forskolin action. These results suggest that forskolin stimulates Na+ transport through a PKA-independent pathway. Furthermore, forskolin increased tyrosine phosphorylation of ∼70- to 80-, ∼97-, and ∼110- to 120-kDa proteins. Protein tyrosine kinase (PTK) inhibitors (tyrphostin A23 and genistein) abolished the forskolin action. Moreover, 5-nitro-2-(3-phenylpropylamino)benzoate (a Cl--channel blocker) prevented the stimulatory action of forskolin on Na+ transport via abolishment of the forskolin-induced cell shrinkage and tyrosine phosphorylation. Based on these results, we conclude that forskolin (and cAMP) stimulates Na+ transport in a PTK-dependent but not a PKA-dependent pathway by causing cell shrinkage, which activates PTK in rat fetal distal lung epithelial cells.

Effects of PKA inhibitors, H-compounds, on epithelial Na+ channels via PKA-independent mechanisms

The Na+ transport in alveolar type II epithelial cells of rat fetal lung was stimulated by cAMP, which is generally thought to act through activation of protein kinase A (PKA). PKA inhibitors (H8, H89 and H7) stimulated amiloride-sensitive Na+ transport in the alveolar type II epithelial cells. H85, an inactive form of H89 as a PKA inhibitor, had also mimicked the stimulatory action of H89 on the Na+ transport. On the other hand, another type of PKA inhibitor, KT5720 or myristoylated PKA inhibitory peptide [14-22] amide, did not stimulate the Na+ transport, but inhibited the Na+ transport unlike H-compounds. These observations suggest that H-compounds act on the Na+ transport depending on the structure.