RESUMEN
Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.
Asunto(s)
Calcio/metabolismo , Músculo Liso/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Tráquea/efectos de los fármacos , Animales , Western Blotting , División Celular/efectos de los fármacos , Células Cultivadas , Masculino , Músculo Liso/fisiología , Fosforilación , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Receptores del Factor de Crecimiento Derivado de Plaquetas/análisis , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Tráquea/fisiologíaRESUMEN
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
Fischer rat airway smooth muscle (ASM) models two potential risk factors for asthma: hyperresponsiveness to contractile agonists and to growth stimuli. The aim of this study was to identify the mechanisms responsible for enhanced ASM mitogenic response in Fischer rats compared with the control Lewis strain. The enhanced Fischer ASM cell growth response to fetal bovine serum (FBS) could not be accounted for by phospholipase C, mitogen-activated protein kinases, or tyrosine kinase activities as assessed by pharmacological inhibition and Western blotting. In contrast, depletion of phorbol ester-sensitive isoforms of the serine/threonine kinase protein kinase C (PKC) removed the difference in growth response between the rat strains. Additionally, FBS selectively induced serine/threonine phosphorylation of a 115-kDa protein in Fischer ASM cells. Enhanced activation of PKC-betaI and decreased activation of PKC-delta in Fischer compared with Lewis cells following FBS stimulation were suggested by Western blotting of membrane and cytosolic fractions. The data are consistent with a role for PKC in the enhanced ASM cell growth of hyperresponsive rats.
Asunto(s)
Bronquios/patología , Hiperreactividad Bronquial/patología , Músculo Liso/patología , Proteína Quinasa C/fisiología , Animales , Bronquios/metabolismo , Bovinos/sangre , Bovinos/embriología , División Celular/fisiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Isoenzimas/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas , Acetato de Tetradecanoilforbol/farmacología , Tirosina/metabolismoRESUMEN
The purpose of this study was to determine if endothelin-1 (ET-1) mediates endothelium-dependent enhancement of rat aortic contractility following exposure to hypoxia. Rats breathed room air or 10% oxygen for 12 or 48 h. Thoracic aortas and plasma were analysed for ET-1 content by radioimmunassay. Aortic rings were mounted in organ bath myographs for measurement of isometric tension during activation by phenylephrine (10(-9)-10(-4) M), in the presence and absence of BQ-123. In some rings, the endothelium was removed. Plasma ET-1 levels were 0.79+/-0.09 pg/ml, 2.00+/-0.36 and 1.88+/-0.21 pg/ml, in normoxic rats and rats exposed to hypoxia for 12 or 48 h respectively (P<0.001, 12 or 48 h vs. control). Aortic ET-1 concentrations were 202.3+/-20.8 fg/mg in normoxic rats, compared to 274.9+/-40.6 fg/mg and 292.4+/-24.4 fg/mg in rats exposed to hypoxia for 12 and 48 h, respectively (P<0.01, 12 or 48 h vs. control) and 155.0+/-43.1 fg/mg in de-endothelialized aortas from rats exposed to hypoxia for 48 h (P>0.05 vs. normoxic controls). Maximum tension during phenylephrine-induced contraction was 0.46+/-0.04 mg/g and 0.33+/-0.03 mg/g in endothelialized rings from rats exposed to hypoxia for 48 h in the presence and absence of BQ-123, respectively (P<0.05 for difference), and 0.28+/-0.07 mg/g in rings in which the endothelium had been removed. Local endothelin release is an important mechanism by which the responsiveness of the systemic vasculature to agonists may be preserved during hypoxia.
Asunto(s)
Endotelina-1/farmacología , Hipoxia/fisiopatología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Endotelio Vascular/fisiología , Masculino , Músculo Liso Vascular/fisiología , Fenilefrina/administración & dosificación , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Vasoconstrictores/administración & dosificación , Vasoconstrictores/farmacologíaRESUMEN
The highly inbred Fisher rat strain demonstrates both hyperresponsiveness of the airways to bronchoconstrictors and increased amounts of airway smooth muscle when compared with Lewis rats. We postulated that the excess airway smooth muscle in Fisher was attributable to a greater sensitivity to endogenous mitogenic stimuli with signaling mechanisms common to contractile stimuli. To test this possibility we investigated differences in the growth response of cultured airway smooth muscle cells (SMC) from these two strains. The Fisher SMC demonstrated an enhanced growth response to fetal bovine serum (FBS), as measured both by cell counting and [3H]thymidine incorporation. The difference in growth response was attributable to a more rapid transition in Fisher than Lewis SMC from the G0/G1 phase to the S phase of the cell cycle. When SMC growth was stimulated by either serotonin, endothelin 1, insulin, platelet-derived growth factor (PDGF), or combinations of the above factors, significant interstrain growth response differences were found only with either human PDGF (AB) or a combination of porcine PDGF (BB) and insulin. A polyclonal antibody to PDGF was less inhibitory to Fisher than Lewis SMC following stimulation with FBS. Thus, interstrain growth differences may reflect different responses to PDGF present in FBS, or to other factors which exert an influence during the G0 or G1 phase of the cell cycle.
Asunto(s)
Ciclo Celular/fisiología , Músculo Liso/citología , Ratas Endogámicas F344/fisiología , Ratas Endogámicas Lew/fisiología , Tráquea/citología , Resistencia de las Vías Respiratorias , Animales , Sangre , Broncoconstrictores/farmacología , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cultivo , Endotelina-1/farmacología , Humanos , Insulina/farmacología , Masculino , Cloruro de Metacolina/farmacología , Mitógenos/farmacología , Músculo Liso/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Ratas , Serotonina/farmacología , Porcinos , Tráquea/efectos de los fármacosRESUMEN
Apamin has been shown to antagonize the nonadrenergic, noncholinergic (NANC) inhibitory system in guinea pig taenia coli. We have examined the effects of apamin on the nonadrenergic noncholinergic inhibitory system and its putative transmitters in isolated guinea pig trachea. Electrical field stimulation (ES) of isolated trachea pretreated with atropine and propranolol evoked reproducible relaxations that were blocked by tetrodoxin, but were unaffected by apamin. Vasoactive intestinal peptide (VIP), adenosine (AD), and adenosine triphosphate (ATP) produced concentration-dependent inhibition of histamine (H)-induced contractions of isolated trachea but the inhibitory actions of these agents were not significantly affected by apamin. In contrast, apamin virtually abolished ES-evoked relaxations in guinea pig isolated taenia caeci, and reduced the inhibition of H-induced contraction by ATP from 40% to 1%. We conclude that neither the NANC inhibitory system in the guinea pig trachea nor its putative mediators VIP, AD, and ATP are antagonized by apamin, in contrast to taenia caeci.
Asunto(s)
Apamina/farmacología , Sistema Nervioso Autónomo/efectos de los fármacos , Venenos de Abeja/farmacología , Músculo Liso/efectos de los fármacos , Adenosina/farmacología , Adenosina Trifosfato/farmacología , Animales , Atropina/farmacología , Estimulación Eléctrica , Femenino , Cobayas , Histamina/farmacología , Técnicas In Vitro , Masculino , Norepinefrina/farmacología , Propranolol/farmacología , Tráquea/efectos de los fármacosRESUMEN
The purpose of the study was to describe the effects of long-term, mild daily exercise on age-associated neuromuscular changes in the Syrian hamster. Daily treadmill exercise (for 26 weeks) was administered throughout a portion (25-37%) of the life span (70-100 weeks), beginning at an age of 4 months, during which period body and muscle weights plateau and subsequently decline. Non-exercised (NEX) animals showed an increase in body weight (20%) until 7 months of age, which subsequently declined with the attainment of late adulthood to values not different from 4-month-old controls (Y) by 10.5 months. Gastrocnemius muscles were atrophied, showed twitch potentiation when stimulated in situ, and contained more type I fibers compared to young controls. Hamsters exercised from 4 to 10.5 months of age (EX) showed elevated body weights, and gastrocnemius muscles showed attenuated atrophy (muscle weight and fiber size), lack of twitch potentiation, and a significantly reduced PFK/CS enzyme ratio. Hamsters exercised only until 7 months were similar to NEX group by 10.5 months of age. Mild daily exercise, maintained throughout adult life into early senescence, attenuates muscle atrophy and promotes adaptive enzymatic changes in atrophying muscles.