RESUMEN
Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.
Asunto(s)
Bradiquinina/fisiología , Señalización del Calcio/fisiología , Músculo Liso/fisiopatología , Tráquea/fisiopatología , Enfermedades de la Tráquea/fisiopatología , Animales , Bradiquinina/farmacología , Calcio/metabolismo , Células Cultivadas , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Contracción Muscular , Músculo Liso/efectos de los fármacos , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/fisiología , Proteína Quinasa C/fisiología , Ratas , Ratas Endogámicas F344/fisiología , Ratas Endogámicas Lew , Factores de Tiempo , Tráquea/efectos de los fármacos , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Airway hyperresponsiveness (AHR) is a phenotype of asthma and can be modeled by the inbred Fisher strain of rat, which is hyperresponsive in vivo relative to the Lewis strain. Enhanced airway smooth muscle (ASM) contractility and Ca(2+) mobilization are associated with the AHR observed in Fisher rats. In this study, we investigated whether the interstrain differences in Ca(2+) mobilization to serotonin (5HT) result from differences in inositol (1,4,5)trisphosphate (IP(3)) metabolism and/or IP(3) receptor (IP(3)R) sensitivity. Ca(2+) mobilization by 5HT in cultured ASM cells from both rat strains was phospholipase C (PLC) dependent. Inositol polyphosphate accumulation, and hence PLC activity, was similar in both rat strains, but a specific IP(3) transient was detectable only in Fisher myocytes in response to 5HT. These findings suggested that IP(3) degradation rather than production differed between the two strains. The Vmax and Michaelis constant (K(m)) of IP(3)-specific 5-phosphatase activity were higher in the particulate fraction of Lewis than in Fisher ASM cell homogenates and appeared to be related to a greater expression of two isoforms of 5-phosphatase (type I and type II) in Lewis cells as shown by Western blot analysis. The sensitivity of the IP(3)R to IP(3) was similar between Fisher and Lewis ASM cells, indicating that the interstrain intracellular Ca(2+) differences were unrelated to IP(3)R function. We propose that interstrain variations in 5-phosphatase activity and expression may give rise to the interstrain differences in IP(3)-mediated Ca(2+) release in ASM and may be a determinant of AHR.
Asunto(s)
Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Músculo Liso/metabolismo , Tráquea/metabolismo , Animales , Técnicas In Vitro , Inositol Polifosfato 5-Fosfatasas , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Serotonina/farmacología , Tráquea/efectos de los fármacos , Tráquea/enzimología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.
Asunto(s)
Señalización del Calcio/fisiología , Músculo Liso/fisiología , Proteínas Tirosina Quinasas/fisiología , Tráquea/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Fosfatos de Inositol/antagonistas & inhibidores , Fosfatos de Inositol/biosíntesis , Isoenzimas/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Proteínas Musculares/metabolismo , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Fosfolipasa C gamma , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Endogámicas F344 , Serotonina/farmacología , Tráquea/citología , Tráquea/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Tirfostinos/farmacologíaRESUMEN
The mechanisms underlying airway hyperresponsiveness are still unknown but increased contractility of airway smooth muscle may play a role. This study sought to demonstrate a relationship between in vivo airway responsiveness and a number of measures of airway smooth muscle responsiveness ex vivo, including intracellular Ca(2+) signaling, by comparing three inbred strains of rat with different degrees of airways responsiveness to methacholine. Lewis, ACI, and Fisher strains of rat were characterized for their pulmonary responses to 5-hydroxytryptamine (5HT) in vivo and Fisher rats were found to be hyperresponsive to 5HT compared with ACI and Lewis rats. The responsiveness of the airways from these strains of rat ex vivo revealed that intraparenchymal airways from Fisher rats significantly narrowed to a greater degree and at a faster rate to 5HT than Lewis rat airways, consistent with their differences in vivo. Intraparenchymal ACI airways, however, narrowed to the same degree as Fisher airways but took longer to do so at a high concentration of 5HT. 5HT caused concentration-dependent increases in intracellular Ca(2+) in airway smooth muscle cells from all three strains of rat, but Fisher and ACI displayed higher responses than Lewis airway smooth muscle. Our results demonstrate that the degree of intracellular Ca(2+) mobilization by 5HT in airway smooth muscle parallels the rate and degree of intraparenchymal airway narrowing and suggest that the degree of intracellular Ca(2+) mobilization plays a role in determining airway smooth muscle contractility.
Asunto(s)
Resistencia de las Vías Respiratorias/fisiología , Hiperreactividad Bronquial/fisiopatología , Calcio/metabolismo , Músculo Liso/fisiopatología , Transducción de Señal/fisiología , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Pruebas de Provocación Bronquial , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/fisiología , Masculino , Cloruro de Metacolina , Músculo Liso/efectos de los fármacos , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Especificidad de la EspecieRESUMEN
The effects of concomitant P1-receptor stimulation on peak intracellular Ca2+ release by extracellular adenosine 5'-triphosphate (ATP) and 5-hydroxytryptamine (5-HT) were investigated in cultured airway smooth-muscle (ASM) cells. The results show that peak Ca2+ release to ATP is enhanced by preincubation with adenosine (ADO) and with the specific A3 receptor agonist 1-Deoxy-1-(6-([(3-iodophenyl)methyl] amino)-9H-purin-9-yl)-N-methyl-beta-D-ribofuranuronamide (1B-MECA). The response to 5-HT, a smooth-muscle contractile agonist, was also enhanced after preincubation with ADO. Further measurements showed that this enhancement of the response to ATP was dependent on extracellular calcium because it was abolished by the removal of Ca2+ from the extracellular fluid and by incubation with the calcium channel blocker nifedipine. In addition, there was no difference between the levels of total inositol phosphates measured in the presence of ATP alone or of ADO + ATP. AACOCF3, a specific blocker of phospholipase A2, decreased the peak Ca2+ response to ATP and abolished the enhanced response to ATP and 5-HT produced by ADO. We conclude that stimulation of P1 and P2 receptors in ASM cells activates not only phospholipase C but also phospholipase A2. The enhancement of ATP-induced and 5-HT-induced Ca2+ release is due to Ca2+ influx from the extracellular fluid through a Ca2+ channel presumably modulated by arachidonic acid. These data show that endogenous ADO may modulate airway hyperresponsiveness by enhancing the ASM response to contractile agonists.