RESUMEN

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]

Asunto(s)

Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/metabolismo , Matemática , Modelos Biológicos , Unión Proteica , Receptores de Droga/agonistas , Receptores de Droga/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas Portadoras/metabolismo , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Colforsina/farmacología , Células Epiteliales/metabolismo , Riñón/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Bumetanida/farmacología , Polaridad Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Epiteliales/citología , Riñón/citología , Nitrobenzoatos/farmacología , Ouabaína/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal , Simportadores de Cloruro de Sodio-Potasio , Tirfostinos/farmacología

RESUMEN

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.

Asunto(s)

Calcio/fisiología , Cloruros/fisiología , Canales Iónicos/fisiología , Alveolos Pulmonares/fisiología , Mucosa Respiratoria/fisiología , Agonistas Adrenérgicos beta/farmacología , Amilorida/farmacología , Animales , Citosol/fisiología , Células Epiteliales/fisiología , Feto/citología , Canales Iónicos/agonistas , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Ratas , Terbutalina/farmacología

RESUMEN

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.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocalasina D/farmacología , Canales Iónicos/metabolismo , Animales , Células Cultivadas , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Xenopus laevis

RESUMEN

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.

Asunto(s)

Agonistas Adrenérgicos beta/farmacología , Cloruros/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Mucosa Respiratoria/enzimología , Sodio/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Tamaño de la Célula/fisiología , Colforsina/farmacología , AMP Cíclico/metabolismo , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Feto/citología , Nitrobenzoatos/farmacología , Embarazo , Ratas , Ratas Wistar , Mucosa Respiratoria/citología , Mucosa Respiratoria/embriología , Tirfostinos/farmacología

RESUMEN

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.

Asunto(s)

Proteínas Tirosina Quinasas/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio , Animales , Transporte Biológico , Línea Celular , Inhibidores Enzimáticos/farmacología , Canales Epiteliales de Sodio , Riñón/citología , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Sodio/metabolismo , Canales de Sodio/fisiología , Tirosina/metabolismo , Tirfostinos/farmacología , Equilibrio Hidroelectrolítico/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Células Epiteliales/metabolismo , Proteínas Quinasas/metabolismo , Estrés Fisiológico/enzimología , Animales , Línea Celular , Tamaño de la Célula , Clorpromazina/farmacología , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrobenzoatos/farmacología , Nistatina/farmacología , Concentración Osmolar , Potasio/farmacología , Xenopus laevis , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

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.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Feto/metabolismo , Pulmón/embriología , Proteínas Tirosina Quinasas/fisiología , Sodio/metabolismo , Amilorida/farmacología , Animales , Transporte Biológico/fisiología , Cationes/metabolismo , Células Cultivadas , Canales de Cloruro/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Feto/citología , Feto/efectos de los fármacos , Feto/fisiología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/efectos de los fármacos , Nitrobenzoatos/farmacología , Fosfotirosina/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Wistar

RESUMEN

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.

Asunto(s)

Carbazoles , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Sulfonamidas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Amilorida/farmacología , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Células Epiteliales/metabolismo , Indoles/química , Indoles/farmacología , Isoquinolinas/química , Isoquinolinas/farmacología , Ácido Mirístico/farmacología , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Pirroles/química , Pirroles/farmacología , Ratas , Ratas Wistar , Sodio/metabolismo , Relación Estructura-Actividad , Terbutalina/farmacología

RESUMEN

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.

Asunto(s)

Canales de Cloruro/fisiología , Células Epiteliales/metabolismo , Riñón/metabolismo , Proteínas Tirosina Quinasas/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Amilorida/farmacología , Animales , Western Blotting , Células Cultivadas , Diuréticos/farmacología , Inhibidores Enzimáticos/farmacología , Soluciones Hipotónicas , Riñón/citología , Nitrobenzoatos/farmacología , Concentración Osmolar , Ouabaína/farmacología , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Tirosina/metabolismo , Xenopus laevis

RESUMEN

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.

Asunto(s)

Calcio/farmacología , Proteínas Portadoras/fisiología , Insulina/farmacología , Canales de Potasio/efectos de los fármacos , Proteínas Tirosina Quinasas/fisiología , Alveolos Pulmonares/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Amilorida/farmacología , Animales , Bumetanida/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Tamaño de la Célula/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Recién Nacido , Fenoles/farmacología , Fosforilación/efectos de los fármacos , Canales de Potasio/fisiología , Embarazo , Embarazo en Diabéticas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Alveolos Pulmonares/metabolismo , Quinina/farmacología , Ratas , Ratas Wistar , Receptor de Insulina/efectos de los fármacos , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatología , Transducción de Señal/fisiología , Canales de Sodio/fisiología , Simportadores de Cloruro de Sodio-Potasio , Tapsigargina/farmacología

RESUMEN

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.

Asunto(s)

Amilorida/farmacología , Calcio/metabolismo , Cloruros/metabolismo , Células Epiteliales/fisiología , Alveolos Pulmonares/fisiología , Canales de Sodio/fisiología , Terbutalina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Células Cultivadas , Citosol/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Feto , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Biológicos , Técnicas de Placa-Clamp , Alveolos Pulmonares/citología , Ratas , Ratas Wistar , Canales de Sodio/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Proteínas Portadoras/efectos de los fármacos , Canales de Cloruro/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Genisteína/farmacología , Isoflavonas/farmacología , Aceites Volátiles/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Portadoras/fisiología , Línea Celular , Membrana Celular/fisiología , Manzanilla , Canales de Cloruro/fisiología , Electrofisiología/métodos , Células Epiteliales , Riñón , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Plantas Medicinales , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Simportadores de Cloruro de Sodio-Potasio , Relación Estructura-Actividad

RESUMEN

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.

Asunto(s)

Calcineurina/metabolismo , Canales de Cloruro/metabolismo , Insulina/fisiología , Riñón/enzimología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Inhibidores de la Calcineurina , Calmodulina/antagonistas & inhibidores , Línea Celular , Canales de Cloruro/efectos de los fármacos , Ciclosporina/farmacología , Citosol , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Isoquinolinas/farmacología , Riñón/metabolismo , Fenoles , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal , Sulfonamidas/farmacología

RESUMEN

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.

Asunto(s)

Cationes/metabolismo , AMP Cíclico/metabolismo , Canales Iónicos/fisiología , Riñón/metabolismo , Sodio/farmacocinética , Sulfonamidas , 1-Metil-3-Isobutilxantina/farmacología , Línea Celular , Citocalasina D/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Canales Iónicos/efectos de los fármacos , Isoquinolinas/farmacología , Riñón/citología , Riñón/efectos de los fármacos , Cinética , Técnicas de Placa-Clamp , Permeabilidad , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Estimulación Física

RESUMEN

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.

Asunto(s)

Agonistas Adrenérgicos beta/farmacología , Calcio/metabolismo , Feto , Pulmón/efectos de los fármacos , Terbutalina/farmacología , Animales , Tamaño de la Célula , AMP Cíclico/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Pulmón/citología , Pulmón/metabolismo , Embarazo , Ratas , Ratas Wistar , Factores de Tiempo

RESUMEN

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.

Asunto(s)

Ciclopentanos/farmacología , Feto/metabolismo , Pulmón/embriología , Sodio/farmacocinética , Terbutalina/farmacología , Absorción/efectos de los fármacos , Amilorida/farmacología , Animales , Brefeldino A , Cationes/metabolismo , Epitelio/embriología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Técnicas de Placa-Clamp , Ratas/embriología , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo

RESUMEN

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 microM, a Ca2+ channel blocker) or 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 microM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 microM 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.

Asunto(s)

1-Metil-3-Isobutilxantina/farmacología , Cloruros/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Sulfonamidas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Canales de Cloruro/antagonistas & inhibidores , AMP Cíclico/metabolismo , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Transporte Iónico , Isoquinolinas/farmacología , Cinética , Nitrobenzoatos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Proteínas Quinasas , Xenopus laevis

RESUMEN

Transforming growth factor-beta (TGF-beta) increases steady-state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF-beta 1 regulation of bone proteins, we have analyzed the effects of the TGF-beta 1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF-beta 1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells approximately 8-fold: the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF-beta 1, and nuclear "run-on" transcription analyses revealed only a approximately 2-fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post-transcriptional mechanism. Moreover, the effects of TGF-beta 1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 micrograms/ml). To identify the site of transcriptional regulation by TGF-beta 1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt -801 to -426 of the promoter sequence were found to enhance transcriptional activity approximately 1.8-fold in cells treated with TGF-beta 1. Within this sequence, approximately 500 nt upstream of the transcription start site, a putative TGF-beta activation element (TAE) was identified that contained the 5'-portion of the nuclear factor-1 (NF-1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein-2 (AP-2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF-beta 1 stimulated cells in gel mobility shift assays and from the attenuation of TGF-beta 1-induced luciferase activity when cells were co-transfected with a double-stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF-1 nuclear protein. These studies have therefore identified a TGF-beta activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF-beta 1 on BSP gene transcription.

Asunto(s)

Regiones Promotoras Genéticas , Sialoglicoproteínas/genética , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Sialoproteína de Unión a Integrina , ARN Mensajero/metabolismo , Ratas , Análisis de Secuencia de ADN , Sialoglicoproteínas/metabolismo , Transfección , Células Tumorales Cultivadas

RESUMEN

We previously demonstrated that histamine and bradykinin evoke an increase in intracellular Ca2+ ([Ca2+]i) in human gingival fibroblasts by using a fluorescent Ca2+ indicator Fura-2. In this paper, we further demonstrate the regulation of the histamine-induced Ca2+ mobilization by bradykinin. In fibroblasts stimulated with bradykinin (1 microM), subsequent stimulation with histamine (100 microM) failed to mobilize Ca2+, whereas bradykinin induced an increase in [Ca2+]i in the cells pre-stimulated with histamine. The attenuation of the histamine response was dependent on the concentration of bradykinin for the first stimulation. Histamine also failed to induce the formation of inositol 1,4,5-trisphosphate in fibroblasts pretreated with bradykinin. In fibroblasts pretreated with bradykinin (1 microM) for 3 min and then washed with fresh medium, the effect of histamine on [Ca2+]i quickly returned to the control level. The activation of protein kinase C by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (PMA) elicited a marked decrease in histamine-induced Ca2+ mobilization. When the protein kinase C activity was inhibited with H7, a protein kinase C inhibitor, or was down-regulated by pretreatment with PMA for 20 h, the inhibitory effect of PMA on the histamine response was relieved. In the fibroblasts pretreated with H7 or PMA for 20 h, histamine evoked Ca2+ mobilization even after bradykinin stimulation. These results suggest that the histamine response is regulated by bradykinin receptor activation via the activation of protein kinase C in human gingival fibroblasts.

Asunto(s)

Bradiquinina/fisiología , Calcio/metabolismo , Encía/metabolismo , Histamina/fisiología , Proteína Quinasa C/metabolismo , Activación Enzimática , Fibroblastos/enzimología , Fibroblastos/metabolismo , Encía/enzimología , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Acetato de Tetradecanoilforbol/farmacología