RESUMEN
Regulation of the increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by bradykinin (BK) led to IP3 formation and caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the BK-induced IP3 formation and Ca2+ mobilization. However, this inhibition was reduced after incubating the cells for 4 h with PMA. Inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses to BK. Prior treatment with staurosporine (1 microM), a PKC inhibitor, inhibited the effect of PMA on the BK-induced response, suggesting that the effect of PMA is mediated by the activation of PKC. In parallel experiments, a change of PKC activity was observed. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the cell membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Moreover, treatment with 1 microM PMA for 2 and 24 h did not significantly change the KD and Bmax of the BK receptor for [H]BK binding (control: KD = 2.3 +/- 0.3 nM, Bmax = 25.2 +/- 1.4 fmol/mg protein). These results suggest that activation of PKC inhibit IP3 accumulation and consequently attenuate [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to BK was associated with an increase in membranous PKC activity.
Asunto(s)
Calcio/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , Receptores de Bradiquinina/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Alcaloides/farmacología , Animales , Bradiquinina/metabolismo , Bradiquinina/farmacología , Células Cultivadas , Perros , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Hidrólisis , Inositol 1,4,5-Trifosfato/biosíntesis , Cinética , Masculino , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Ésteres del Forbol/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Estaurosporina , TráqueaRESUMEN
The effect of forskolin on 5-hydroxytryptamine (5-HT)-induced inositol phosphate (IP) and Ca2+ mobilisation was investigated in canine cultured aorta smooth muscle cells (ASMCs). Pretreatment of ASMCs with forskolin attenuated 5-HT-induced IP accumulation and Ca2+ mobilisation in a time- and concentration-dependent manner. The half-maximal effects (pEC50) of forskolin to attenuate IP and Ca2+ responses to 5-HT occurred at concentrations of 6.28 and 6.64, respectively. Pretreatment of ASMCs with cholera toxin caused a similar inhibition on 5-HT-induced responses. Even after treatment with forskolin for 24 h, the 5-HT-induced responses were still inhibited. The inhibitory effect of forskolin resulted from both a depression of the maximal response and a shift to the right of the concentration-effect curves of 5-HT in these responses. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated IP accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on IP response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-iosquinolinesulphonamide] and HA-1004 [N-(2-guanidinoethyl)-5-iosquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IP in ASMCs. These results indicate that activation of cAMP/PKA might inhibit the 5-HT-stimulated IP accumulation and consequently reduce Ca2+ mobilisation, or inhibit both responses independently.
Asunto(s)
Calcio/metabolismo , Colforsina/farmacología , Músculo Liso Vascular/metabolismo , Fosfatidilinositoles/metabolismo , Serotonina/farmacología , Animales , Aorta/citología , Células Cultivadas , Toxina del Cólera , Colforsina/metabolismo , Perros , Femenino , Hidrólisis , Fosfatos de Inositol/metabolismo , Masculino , Músculo Liso Vascular/citología , Antagonistas de la Serotonina/farmacologíaRESUMEN
Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.
Asunto(s)
Serotonina/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/metabolismo , Animales , Aorta/citología , Calcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Perros , Inhibidores Enzimáticos/farmacología , Fosfatos de Inositol/metabolismo , Isoenzimas/metabolismo , Músculo Liso Vascular/citología , Proteína Quinasa C/antagonistas & inhibidores , Serotonina/farmacología , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on bradykinin (BK)-induced generation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with either forskolin or dibutyryl cyclic AMP attenuated BK-stimulated responses. The inhibitory effects of these agents produced both a depression of the maximal response and a shift to the right of the concentration-response curves of BK. The water-soluble forskolin analogue L-858051, 7-deacetyl-7 beta-(r-N-methylpiperazino)-butyryl forskolin, significantly attenuated BK-stimulated IPs accumulation, while 1,9-dideoxy forskolin, an inactive forskolin, had little effect on IPs response. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cyclic AMP-dependent protein kinase (PKA), reversed the ability of forskolin to attenuate BK-stimulated IPs accumulation. The KD and Bmax, values of the BK receptor for [3H]BK binding were not significantly changed by forskolin treatment for 30 min and 4 h. The AlF4(-)-induced IPs accumulation was attenuated by forskolin, indicating that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. These results suggest that activation of cyclic AMP/PKA might inhibit the BK-stimulated PI breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses, which is distal to the BK receptor in canine cultured TSMCs.
Asunto(s)
Bradiquinina/farmacología , Calcio/metabolismo , Colforsina/farmacología , Músculo Liso/metabolismo , Tráquea/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/fisiología , Compuestos de Aluminio/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Bradiquinina/metabolismo , Bucladesina/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Colforsina/análogos & derivados , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Perros , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Fluoruros/farmacología , Fosfatos de Inositol/metabolismo , Masculino , Receptores de Bradiquinina/metabolismo , Saponinas/farmacología , Transducción de Señal/fisiología , Tráquea/citologíaRESUMEN
The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.
Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Fosfatidilinositoles/metabolismo , Serotonina/metabolismo , Animales , Aorta/citología , Células Cultivadas , Perros , Femenino , Hidrólisis , Fosfatos de Inositol/metabolismo , Masculino , Músculo Liso Vascular/citología , Toxina del Pertussis , Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
5-Hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) was monitored in cultured canine tracheal smooth muscle cells (TSMCs) using a fluorescent Ca2+ indicator Fura-2. Stimulation of TSMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation of [Ca2+]i. The log (EC50) values of 5-HT for the peak and sustained plateau responses were -7.43 and -7.60 M, respectively. 5-HT1A and 5-HT3 receptor antagonists, NAN-190 and metoclopramide, inhibited the 5-HT-stimulated increase in [Ca2+]i with pKB values of 6.3 and 6.2, respectively, indicating that the 5-HT receptors mediating Ca2+ signal had low affinity for these receptor antagonists. In contrast, 5-HT2A receptor antagonists, ketanserin and mianserin, had high affinity in antagonizing the changes in [Ca2+]i response to 5-HT with pKB values of 8.3 and 8.3, respectively. The sustained elevation of [Ca2+]i was dependent on the presence of extracellular Ca2+. Removal of extracellular Ca2+ by addition of 2 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to the resting level. In the absence of extracellular Ca2+, only an initial peak was observed which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to 5-HT. These Ca(2+)-channel blockers also decreased the sustained elevation of [Ca2+]i when applied during the plateau phase. In conclusion, these findings indicate that the initial increase in [Ca2+]i stimulated by 5-HT acting on 5-HT2A receptors is due to the release of Ca2+ from internal stores, followed by the influx of external Ca2+ into the cells. The influx of extracellular Ca2+ partially involves a diltiazem and verapamil sensitive Ca2+ channel.
Asunto(s)
Calcio/metabolismo , Músculo Liso/metabolismo , Serotonina/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Perros , Femenino , Fura-2 , Masculino , Músculo Liso/efectos de los fármacos , Antagonistas de la Serotonina/farmacología , Tráquea/metabolismoRESUMEN
The effects of increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The cAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) at concentrations of 7.6 +/- 1.3 g/ml and 4.8 +/- 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca2+]i with half-maximal inhibition (-logEC50) at 5.2 +/- 0.7 M. Pretreatment of TSMCs with either CTX (10 micrograms/ml, 4 h), forskolin (10-100 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca2+ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC50 values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca2+ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 microM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 microM], an inhibitor of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The KD and Bmax values of the muscarinic receptor (mAChR) for [3H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF4(-)-stimulated IPs accumulation in canine TSMCs. The AIF4(-)-induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF4- and uncoupled to phospholipase C by forskolin treatment. We conclude that cAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle formation.
Asunto(s)
Calcio/metabolismo , Carbacol/farmacología , AMP Cíclico/fisiología , Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , Sulfonamidas , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Compuestos de Aluminio/farmacología , Animales , Bucladesina/farmacología , Células Cultivadas , Toxina del Cólera/farmacología , Colforsina/farmacología , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Perros , Inhibidores Enzimáticos/farmacología , Femenino , Fluoruros/farmacología , Proteínas de Unión al GTP/fisiología , Hidrólisis , Isoquinolinas/farmacología , Masculino , Músculo Liso/citología , Fosfatidilinositoles/biosíntesis , Receptores Muscarínicos/metabolismo , Transducción de Señal/fisiología , TráqueaRESUMEN
Regulation of the increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP3 formation and caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the ET-1-induced IP3 formation and Ca2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-logEC50) of ET-1-induced IP3 formation and increase in [Ca2+]i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the ET-1-induced IP3 formation and Ca2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.
Asunto(s)
Calcio/metabolismo , Endotelinas/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Músculo Liso/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Tráquea/metabolismo , Alcaloides/farmacología , Animales , Células Cultivadas , Diglicéridos/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Interacciones Farmacológicas , Hidrólisis , Contracción Muscular/fisiología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Transducción de Señal , Estaurosporina , Tráquea/citología , Tráquea/efectos de los fármacosRESUMEN
1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the 5-HT-induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca2+ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 microM) of GF109203X (0.1 microM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP3 formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-alpha, beta I, beta II, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down-regulation of these PKC isozymes PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta induced by PMA caused an attenuation of 5-HT-stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.
Asunto(s)
Calcio/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , Antagonistas de la Serotonina/farmacología , Serotonina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Animales , Western Blotting , Células Cultivadas , Perros , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidrólisis , Fosfatos de Inositol/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Tráquea/citología , Tráquea/efectos de los fármacos , Tráquea/metabolismoRESUMEN
Regulation of the increases in inositol 1,4,5-trisphosphate (IP(3)) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP(3) formation and caused an initial transient peak of [Ca(2+)](i) followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 &mgr;M) for 30 min blocked the carbachol-induced IP(3) formation and Ca(2+) mobilization. Following preincubation, carbachol-induced Ca(2+) mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP(3) formation and increase in [Ca(2+)](i) were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 &mgr;M), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 &mgr;M, did not inhibit these responses to carbachol. The K(d) and B(max) of the muscarinic receptor for [(3)H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity. Copyright 1995 S. Karger AG, Basel
RESUMEN
Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca2+ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca2+]i, with half-maximal effect (EC50) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ETA receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca2+ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca2+]i to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i response to these peptides. ETs exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization of the Ca2+ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca2+ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by these peptides is due to the activation of ETA receptors and subsequently the release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Endotelinas/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Vasoconstrictores/farmacología , Venenos de Víboras/farmacología , Animales , Carbacol/farmacología , Células Cultivadas , Diltiazem/farmacología , Perros , Antagonistas de los Receptores de Endotelina , Femenino , Masculino , Péptidos Cíclicos/farmacología , Tráquea/efectos de los fármacos , Verapamilo/farmacologíaRESUMEN
The effects of increases in cellular adenosine 3'5'-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) produced at concentrations of 7.0 +/- 0.5 and 4.9 +/- 0.4 respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca2+ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF4--induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF4-- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca2+]i increase or inhibit both responses independently.