Photodynamic therapy with acridine orange in musculoskeletal sarcomas.

Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. We have been studying alternative treatments for musculoskeletal sarcoma since 1990, and have recently established a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells. These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

New concept of spare receptors and effectors.

The present study provides a new concept of the spare receptor. Model [A]: 1) Several receptors connect with an effector; 2) if an agonist occupies one of the receptors connecting with one effector, the effector fully functions. When the number of receptors connecting with one effector is "m", the relationship between the functional effectors (E) and the concentration of agonists ([a]) is as follows: [formula: see text] where Rt is the total number of receptors and Kd is the agonist dissociation constant from the receptor. Model [B]: 1) Several receptors connect with an effector; 2) only when agonists occupy all of the receptors connecting with one effector, the effector functions. The relationship between E and [a] is as follows: [formula: see text] If m=1, equations (I) and (II) are exactly the same as the Michaelis-Menten equation. If m is larger than 1, the apparent saturation in the effector efficiency becomes larger in Model [A], and smaller in Model [B], respectively. The dissociation of the fractional efficiency of effectors from the fractional binding of agonists to receptors becomes larger as m becomes larger in both models. Further, we propose a variable model, including the concept of agonist-occupancy-dependent stability in the functional conformation change of the effector; only when more than j pieces of receptors connecting with one effector are occupied by agonists, the effector functions (Model [M]). The relationship between E and [a] is as follows: [formula: see text]

Forskolin activation of apical Cl- channel and Na+/K+/2Cl- cotransporter via a PTK-dependent pathway in renal epithelium.

Forskolin induced the transepithelial Cl- transport (secretion) by activating the apical Cl- channel and basolateral Na+/K+/2Cl- cotransporter in renal epithelial A6 cells via an increase in cytosolic cAMP concentration. The cAMP activation of apical Cl- channel and Na+/K+/2Cl- cotransporter was partially mediated through a protein kinase A (PKA)-dependent pathway, but a PKA-independent pathway was also suggested to be involved in the cAMP activation. Therefore, we assessed a possibility of involvement of protein tyrosine kinase (PTK)-dependent pathway as a PKA-independent pathway in the cAMP activation by applying a PTK inhibitor, tyrphostin A23 (AG18). Tyrphostin A23 abolished the forskolin-induced transepithelial Cl- secretion by partially diminishing the activity of the Cl- channel and completely inhibiting the Na+/K+/2Cl- cotransporter. Further, forskolin increased phosphorylation of protein tyrosine, suggesting that cAMP activates PTK. These observations suggest that cAMP activates the Cl- channel and the Na+/K+/2Cl- cotransporter by activating PTK.

The essential role of cytosolic Cl- in Ca2+ regulation of an amiloride-sensitive channel in fetal rat pneumocyte.

An amiloride-sensitive, Ca(2+)-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na+ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca2+ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (Po) of the NSC channel to 0.62 +/- 0.07 from 0.03 +/- 0.01 (mean +/- SE; n = 8) 30 min after application of terbutaline in a solution containing 1 mM Ca2+. The Po of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca2+ to 0.26 +/- 0.05 (n = 8). The cytosolic Ca2+ concentration ([Ca2+]c) in the presence and absence of extracellular Ca2+ was, respectively, 100 +/- 6 and 20 +/- 2 nM (n = 7) 30 min after application of terbutaline. The cytosolic Cl- concentration ([Cl-]c) in the presence and absence of extracellular Ca2+ was, respectively, 20 +/- 1 and 40 +/- 2 mM (n = 7) 30 min after application of terbutaline. The diminution of [Ca2+]c from 100 to 20 nM itself had no significant effects on the Po if the [Cl-]c was reduced to 20 mM; the Po was 0.58 +/- 0.10 at 100 nM [Ca2+]c and 0.55 +/- 0.09 at 20 nM [Ca2+]c (n = 8) with 20 mM [Cl-]c in inside-out patches. On the other hand, the Po (0.28 +/- 0.10) at 20 nM [Ca2+]c with 40 mM [Cl-]c was significantly lower than that (0.58 +/- 0.10; P < 0.01; n = 8) at 100 nM [Ca2+]c with 20 mM [Cl-]c' suggesting that reduction of [Cl-]c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca2+ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl-]c.

Blocking action of cytochalasin D on protein kinase A stimulation of a stretch-activated cation channel in renal epithelial A6 cells.

We have shown that the apical membrane of renal epithelial A6 cells has a 29-pS stretch-activated nonselective cation (NSC) channel, which is activated by cytosolic cyclic AMP (cAMP) (J Gen Physiol 1997;110:327-36). In general, downstream signalings of cAMP are mediated through a cAMP-activated protein kinase (protein kinase A, PKA)-dependent pathway. Therefore, to study if the channel is activated by a PKA-dependent pathway, we applied a PKA catalytic subunit directly to the channel from the cytosolic surface in cytosol-free excised inside-out patches, using the single channel recording (patch clamp) technique. Application of PKA catalytic subunit with 2 mM ATP increased the open probability (P(o)) of the channel from 0.11 +/- 0.04 to 0.58 +/- 0.10 (mean +/- SD, N = 11, P < 0.001). The channel has a gating kinetics "C(L) <--> C(S) <--> O, " where C(L,) C(S,) and O are the long closed state, the short closed state, and the open state, respectively. PKA influenced the communication of the channel between C(L) and C(S) without affecting the communication between C(S) and O, leading the channel to only stay in C(S) and O. The PKA-induced increase in P(o) was attributable to the interruption of communication between C(L) and C(S) or to the reduction of time the channel stays in C(L.) Pretreatment with cytochalasin D (Cyt-D), an inhibitor of the polymerization of actin filaments, blocked the stimulatory effect of PKA on the channel. These observations suggest that phosphorylation of polymerized actin filaments regulates the gating kinetics of a stretch-activated channel in renal epithelium.

Beta agonist regulation of sodium transport in fetal lung epithelium: roles of cell volume, cytosolic chloride and protein tyrosine kinase.

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.

Involvement of protein tyrosine kinase in osmoregulation of Na(+) transport and membrane capacitance in renal A6 cells.

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, I(Na)) by decreasing the elevating rate of the hyposmolality-increased I(Na). 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 I(Na) by diminishing the elevating rate of the hyposmolality-increased I(Na), 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.

Cell swelling activates stress-activated protein kinases, p38 MAP kinase and JNK, in renal epithelial A6 cells.

Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl(-) channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K(+) iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells.

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 beta-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 approximately 70- to 80-, approximately 97-, and approximately 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.

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.

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.

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 microm, an inhibitor of Na+/K+/2Cl- cotransporter) and amiloride (10 microm, 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 microm) or thapsigargin (1 microM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 microm, 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.

Regulation of an amiloride-sensitive Na+-permeable channel by a beta2-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 beta2-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 Cl- channel and Na+/K+/2Cl- cotransporter in renal epithelial A6 cells by flavonoids: genistein, daidzein, and apigenin.

The present study investigates regulation of Cl- channels and Na+/K+/2Cl- cotransporter in a renal epithelial cell line, A6, by flavones: genistein [an inhibitor of protein tyrosine kinases (PTK)], daidzein (an inactive compound of genistein), and apigenin [an inhibitor of mitogen-activated protein (MAP) kinase]. Genistein and daidzein activated Cl- channels. Genistein and apigenin had a stimulatory effect on the bumetanide-sensitive Na+/K+/2Cl- cotransporter. Other PTK inhibitors, tyrphostin A23, lavendustin A, and herbimycin A, which do not contain a structure flavone, had no stimulatory action on Cl- channels or the Na+/K+/2Cl- cotransporter. These observations conclude that (i) genistein activates a Cl- channel and the Na+/K+/2Cl- cotransporter; and (ii) the stimulatory action is not mediated through its inhibitory action on protein tyrosine kinase, but rather the structure of flavone itself plays a crucial role in stimulatory regulation of Cl- channels and Na+/K+/2Cl- cotransporter.

Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+.

The epithelial Na+ channel (ENaC), composed of three subunits (alpha, beta, and gamma), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat alpha beta gamma ENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the alpha beta gamma rENaC-expressing MDCK cells exhibited greater whole cell Na+ current at -143 mV (-1,466.2 +/- 297.5 pA) than did untransfected cells (-47.6 +/- 10.7 pA). This conductance was completely and reversibly inhibited by 10 microM amiloride, with a Ki of 20 nM at a membrane potential of -103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing alpha beta or alpha gamma subunits alone was -115.2 +/- 41.4 pA and -52.1 +/- 24.5 pA at -143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-D-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be approximately 5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (Po), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nPo) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (microM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 microM Ca2+ caused a biphasic inhibition, with time constants of 1.7 +/- 0.3 min (n = 3) and 128.4 +/- 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 microM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 microM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 microM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.

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 (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.

Protein phosphatase 2B-dependent pathway of insulin action on single Cl- channel conductance in renal epithelium.

The apical membrane of distal nephron epithelium (A6) has a Ca(2+)-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 phosphoserine/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.

Activation of Na+-permeant cation channel by stretch and cyclic AMP-dependent phosphorylation in renal epithelial A6 cells.

It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl- (PCl/PNa < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (Po) of the IBMX-activated channel. This channel had one open ("O") and two closed (short "CS" and long "CL") states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the Po of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

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

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 beta2-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 microM) from basolateral side; this response was abolished by pretreatment with brefeldin A (1 microg/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 beta2-specific adrenergic agonists promote the trafficking of amiloride sensitive Na+-permeable nonselective cation channels to the apical cell surface.

Beta 2-agonist regulation of cell volume in fetal distal lung epithelium by cAMP-independent Ca2+ release from intracellular stores.

The effects of beta 2-adrenoceptor agonist (beta 2 agonist) and cAMP on cytosolic Ca2+ concentration ([Ca2+]c) and cell volume were studied in fetal distal lung epithelial cells. Both terbutaline (a specific beta 2 agonist, 10 microM) and dibutyryl cAMP (DBcAMP, 1 mM) increased [Ca2+]c in the presence of extracellular Ca2+. Even in the absence of extracellular Ca2+, the terbutaline-induced increase in [Ca2+]c was still observed, although the increase was transient. However, DBcAMP caused no significant change in [Ca2+]c. In the presence of 1 mM extracellular Ca2+, terbutaline and DBcAMP induced quinine (a blocker of K+ channel) sensitive cell shrinkage. However, in a Ca2(+)-free solution, terbutaline induced rapid cell shrinkage, followed by benzamil (a specific blocker of Na+ channel, an analogue of amiloride) sensitive transient cell swelling. In a Ca2(+)-free solution, DBcAMP induced benzamil-sensitive transient cell swelling without cell shrinkage. Taken together, our observations indicate that the beta 2 agonist induced an elevation of [Ca2+]c by increasing both a Ca2+ influx from the extracellular space and a Ca2+ release from intracellular Ca2+ stores, whereas DBcAMP only stimulated Ca2+ influx from the extracellular space. Furthermore, it is suggested that terbutaline and DBcAMP activated benzamil-sensitive channels independently of an increase in [Ca2+]c.