Yoshinori Marunaka

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.

Amiloride-blockable Ca2+-activated Na+-permeant channels in the fetal distal lung epithelium.

The Na+ transport function of alveolar epithelium represents an important mechanism for clearance of fluid in air space at birth. I observed the activity of two types of amiloride-blockable Na+-permeant cation channels in the apical membrane of fetal distal lung epithelium cultured on permeable filters for 2 days after harvesting of the cells from Wistar rats of 20 days gestation (term = 22 days). One type was a nonselective cation (NSC) channel and had a linear current/voltage (I/V) relationship with a single-channel conductance of 26.9 ± 0.8 pS (n = 5). The other type was highly Na+ selective (i.e. Na+ channel) and had an inwardly rectifyingI/V relationship with a single-channel conductance of 11.8 ± 0.2 pS (n = 5) around resting membrane potential. The NSC channel was more frequently observed (1.37 ± 0.15 per patch membrane;n = 73) than the Na+ channel (0.15 ± 0.40 per patch membrane;n = 73). However, the open probability of the NSC channel was smaller than that of the Na+ channel. Both types of the channels were activated by cytosolic Ca2+, however the sensitivity to cytosolic Ca2+ was much higher in the Na+ channel than in the NSC channel. Furthermore, both types of the channels were blocked by amiloride or benzamil. The half-maximal inhibitory concentration (IC50) of amiloride or benzamil of the Na+ channel was 1–2 μM, while that of NSC channel was less than 1 μM. Both channels were activated by insulin.

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( I sc) without affecting total I sc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the I sc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

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.

Cytosolic Ca2+-induced modulation of ion selectivity and amiloride sensitivity of a cation channel and beta agonist action in fetal lung epithelium

The cytosolic Ca2+ concentration ([Ca2+]i) affects many cell functions, including the modulation of ion channel activity. In this study patch clamp experiments using primary cultures of fetal distal lung epithelium (FDLE) demonstrated that the elevation of [Ca2+]i modulated a 25pS amiloride-blockable non selective cation (NSC) channels ion selectivity and sensitivity to amiloride. An elevation of [Ca2+]i from 0.1 microM to 1mM both increased open probability (Po) and decreased the ratio of the permeability to Na to the permeability to K (PNa/PK) from 1.96 +/- 0.11 (SEM, n = 6) to 0.88 +/- 0.04 (n = 6). Within the range of [Ca2+]i from 0.1 microM to 100 microM amiloride (0.5 microM) decreased Po, however amiloride (0.5 microM) no longer affected Po of the NSC channel when [Ca2+]i was increased to 1mM under physiologic membrane potentials. A beta adrenergic agonist (terbutaline, 10 microM) increased Po in cell-attached patches from almost 0 (Po less than 0.01; n = 9) to 0.39 +/- 0.09 (n = 9) and [Ca2+]i from 40 +/- 6nM (n = 9) to more than 1 microM. This suggested that amiloride-blockable NSC channel activity and ion permeability are modulated by changes in [Ca2+]i near physiologic membrane potentials and a beta adrenergic agonist increases [Ca2+]i to more than 1 microM (unlike other epithelial including adult alveolar cells) which is associated with activation the NSC channel.

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

Abstractu2002We 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.

Abstractu2002We 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.

Regulation of cell volume by beta2-adrenergic stimulation in rat fetal distal lung epithelial cells.

Abstract. Cell-volume changes induced by terbutaline (a specific β2-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively differed with the concentration of terbutaline. Terbutaline of 10−10–10−8m induced transient cell swelling. Terbutaline of 10−7m induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10−6–10−5m induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10−3m induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10−6m suppressed the cell swelling induced by 10−10–10−8m terbutaline and quinine of 10−3m inhibited the cell shrinkage induced by 10−6–10−5m terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux. Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10−9–10−3m): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10−4m), like terbutaline (10−5m), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 × 10−10m ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca2+ abolished the cell swelling caused by terbutaline of 10−10–10−8m. With removal of extracellular Ca2+, the initial, rapid cell shrinkage induced by 10−5m terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular Ca2+. Overall, at low concentrations (10−10–10−8m), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca2+-dependent; high concentrations (≥−6) induced quinine-sensitive rapid cell shrinkage, which was Ca2+-dependent; high concentrations (≥−7) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in FDLE cells by cytosolic cAMP and Ca2+ through activation of Na+ and K+ channels.

Effects of vasopressin on single Cl− channels in the apical membrane of distal nephron cells (A6)

We have investigated how two types of Cl- channels found in sodium transporting epithelium are regulated by arginine vasopressin (AVP). A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl- channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without AVP pretreatment, the 3 pS Cl- channel was more frequently observed than the 8 pS Cl- channel. AVP increased the open probability (Po) and single channel conductance of the 3 pS Cl- channel without significantly changing the Po or conductance of the 8 pS Cl- channels. On the other hand, AVP did not affect the number of the 3 pS Cl- channel, but increased the number of 8 pS Cl- channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., (1) increases the Po and single channel conductance of 3 pS Cl- channels and (2) increases the number of 8 pS Cl- channels.