RESUMEN
It was previously found that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs (1-31). In the present study, human plasma concentrations of ET-1 (1-31) and ET-1 were examined and the effect of synthetic ET-1 (1-31) on the proliferation of cultured human mesangial cells (HMCs) was investigated. The proliferative effect of ET-1 (1-31) was evaluated from the [3H]-thymidine uptake. The activity of extracellular signal-regulated kinase (ERK) and DNA binding activity of activator protein-1 were determined by using an in-gel kinase assay and gel mobility shift assay, respectively. Immunoreactive ET-1 (1-31) was detectable in plasma, but the level was slightly lower than that of ET-1. ET-1 (1-31) increased [3H]-thymidine incorporation in HMCs to a degree similar to that induced by ET-1. ET-1 (1-31) also activated ERK1/2. Inhibition of protein kinase C and ERK kinase caused a reduction of ET-1 (1-31)-induced ERK1/2 activation. The ERK1/2 activation was followed by an increase in transcription factor activator protein-1 DNA binding activity. These findings suggest that ET-1 (1-31) is a bioactive peptide in humans and ET-1 (1-31) itself stimulates HMC proliferation.
Asunto(s)
Endotelinas/farmacología , Mesangio Glomerular/efectos de los fármacos , Quinasa 1 de Quinasa de Quinasa MAP , Fragmentos de Péptidos/farmacología , Adulto , División Celular/efectos de los fármacos , Células Cultivadas , ADN/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Antagonistas de los Receptores de Endotelina , Endotelina-1/farmacología , Endotelinas/sangre , Endotelinas/farmacocinética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Glicopéptidos/farmacología , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/farmacocinética , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismoRESUMEN
Protein kinase C (PKC) family, is now classified into three groups; conventional (cPKC), novel (nPKC) and atypical (aPKC), and to date, 10 members of isozymes have been identified. We have suggested that PKC is essential to interleukin-1 (IL-1)-triggered expression of inducible NO synthase (iNOS), and that by pharmacological analysis, cPKC is not involved in iNOS induction in rat vascular smooth muscle cells (VSMC). In the present study, we identified some PKC isozymes and investigated the effect of PKC alpha knockdown by antisense oligodeoxynucleotide (AS-ODN) strategy on iNOS expression and nuclear translocation of NF-kappa B in RASMC. Western blot analysis revealed the presence of cPKC (alpha), nPKCs (delta and epsilon) and aPKCs (tau and lambda). Short-time (10-20 min) treatment with phorbol 12-myristate 13-acetate (PMA) induced translocation of PKC alpha from cytosolic to particulate fraction. PKC alpha was completely downregulated by treatment with 100 nM PMA for 24 hours. Treatment with AS-ODN against PKC alpha mRNA depleted PKC alpha specifically, and had no detectable effect on the other PKCs. The production of iNOS mRNA, but not nuclear translocation of NF-kappa B, stimulated by IL-1 beta was decreased by PKC alpha knockdown. These results suggest that there are 5 PKC isozymes in RASMC, and that PKC alpha is involved in iNOS expression triggered by IL-1 beta, supporting our previous pharmacological conclusion.
Asunto(s)
Interleucina-1/fisiología , Isoenzimas/fisiología , Óxido Nítrico Sintasa/análisis , Oligonucleótidos Antisentido/farmacología , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C/fisiología , Animales , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/aislamiento & purificación , Inducción Enzimática , Isoenzimas/aislamiento & purificación , Masculino , Músculo Liso Vascular/citología , FN-kappa B/genética , Óxido Nítrico Sintasa de Tipo II , Proteína Quinasa C-alfa , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
1. We investigated the effect of SK&F96365, a putative inhibitor of receptor-operated Ca2+ entry, on the endothelium-dependent, NO-mediated relaxation and cyclic GMP formation induced by Ca2(+)-ATPase inhibitors in rat thoracic aorta. 2. SK&F96365 inhibited cyclopiazonic acid or thapsigargin-induced relaxation and cyclic GMP formation mediated by a constitutive NO synthase, which is known to be activated by the Ca2+ that enters into the endothelial cells via plasma membrane Ca2+ channels subsequent to depletion of stored Ca2+ by Ca2(+)-ATPase inhibitors. 3. SK&F96365 also inhibited relaxation and cyclic GMP formation induced by acetylcholine, without affecting those induced by nitroprusside and A23187. 4. Ni2+ attenuated relaxation and cyclic GMP formation induced by cyclopiazonic acid and acetylcholine. 5. In contrast, the voltage-dependent Ca2+ channel blocker, nifedipine, did not affect the relaxation caused by Ca2(+)-ATPase inhibitors. 6. These results suggest that endothelium-dependent, NO-mediated relaxation of the arteries induced by Ca2(+)-ATPase inhibitors is triggered by the Ca2+ that enters into endothelial cells via receptor-operated channels (SK&F96365-sensitive channels) subsequent to depletion of stored Ca2+ as a result of inhibition of the Ca2(+)-ATPase (Ca2+ pump) of the stores.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Endotelio Vascular/efectos de los fármacos , Imidazoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/antagonistas & inhibidores , Animales , Aorta Torácica/metabolismo , Calcio/metabolismo , GMP Cíclico/biosíntesis , Endotelio Vascular/metabolismo , Técnicas In Vitro , Indoles/farmacología , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Níquel/farmacología , Óxido Nítrico/fisiología , Ratas , Ratas Wistar , Terpenos/farmacología , TapsigarginaRESUMEN
We investigated the effect of triptoquinone-A (TQA), an active principal of Triptergium wilfordii, on the induction of nitric oxide synthase (NOS) promoted by endotoxin (LPS) and interleukin-1 beta (IL-1 beta). Prophylactic application of TQA selectively prevented LPS-primed initiation of L-arginine (Arg)-induced relaxation, and cGMP formation of rat thoracic aorta, and LPS-stimulated nitrite production by cultured aortic smooth muscle cells, which appear to be mediated by NOS expressed by LPS in vascular smooth muscle. TQA also prevented IL-1 beta-triggered initiation of Arg-induced relaxation and nitrite accumulation. These results suggest that TQA prevents LPS-or IL-1 beta-promoted induction of NOS in vascular smooth muscle, thus inhibiting development of Arg-induced vasorelaxation.
Asunto(s)
Aorta/efectos de los fármacos , Arginina/farmacología , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Medicina Tradicional China , Óxido Nítrico Sintasa/efectos de los fármacos , Animales , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas WistarRESUMEN
We have suggested that cyclopiazonic acid (CPA), a Ca(2+)-ATPase inhibitor, produces nitric oxide (NO) by triggering a Ca(2+)-influx resulting from Ca(2+)-depletion in the endoplasmic reticulum of endothelial cells. The tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate, while having no effect on relaxations induced by A23187 or Na nitroprusside, did inhibit the CPA-induced relaxation and cyclic GMP formation in rat aorta. Tyrosine kinase may participate in endothelial NO synthesis through activation of a Ca2+ entry mechanism.
Asunto(s)
Calcio/metabolismo , Óxido Nítrico/metabolismo , Proteínas Tirosina Quinasas/farmacología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Indoles/farmacología , Masculino , Nitroprusiato/farmacología , Ratas , Ratas WistarRESUMEN
Tyrosine kinase inhibitors herbimycin A, genistein and erbstatin analog prevented endotoxin (LPS)-promoted initiation of L-arginine (Arg)-induced relaxations and cGMP formation in rat thoracic aorta, which appear to be mediated by nitric oxide synthase expressed by LPS in the vascular smooth muscle. Similarly, interleukin-1 beta (IL-1 beta) triggered initiation of Arg-induced relaxation of the arteries. In addition, in the aortic smooth muscle cells cultured in the presence of Arg, LPS- or IL-1 beta-triggered accumulation of nitrite was suppressed by the tyrosine kinase inhibitors. These results suggest that tyrosine kinase is involved in the LPS- and IL-1 beta-promoted induction of nitric oxide synthase in the vascular smooth muscle, which in turn mediates production of NO from added Arg, thus stimulating formation of cGMP and causing relaxation. Alternatively, it is possible that LPS acts indirectly through cytokines such as IL-1 beta.
Asunto(s)
Aminoácido Oxidorreductasas/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Vasodilatación/efectos de los fármacos , Animales , Aorta Torácica , Arginina/farmacología , Benzoquinonas , Células Cultivadas , Dactinomicina/farmacología , Endotelio Vascular/fisiología , Inducción Enzimática/efectos de los fármacos , Genisteína , Hidroquinonas/farmacología , Técnicas In Vitro , Isoflavonas/farmacología , Lactamas Macrocíclicas , Lipopolisacáridos/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Ratas , Rifabutina/análogos & derivadosRESUMEN
1 The effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 microM, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA-induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG-nitro L-arginine (3-300 microM), the free radical scavenger haemoglobin (0.1-3 microM), the soluble guanylate cyclase inhibitor, LY83583 (0.1-10 microM), each inhibited the endothelium-dependent relaxation to CPA. The potassium channel blocker, glibenclamide (10 microM) and cyclo-oxygenase inhibitor, indomethacin (100 microM for 60 min and then washed out) did not alter the action of CPA. 4 The calmodulin inhibitors calmidazolium (3-10 microM) and W-7 (100 microM) also abolished CPA-induced relaxation. 5 CPA (10 microM) increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in arteries with an intact endothelium, without affecting adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 6 The inhibitors of NO synthesis and actions, the calmodulin inhibitor and removal of the endothelium abolished the CPA-stimulated increase in the levels of cyclic GMP. 7 In Ca(2+)-free solution, CPA failed to induce relaxation or to stimulate cyclic GMP production. Relaxation to nitroprusside was not affected under these conditions. 8 These results suggest that CPA can stimulate NO synthesis, possibly by inhibiting a Ca(2+)-ATPase, which replenishes Ca2+ in the intracellular storage sites in endothelial cells. Depletion of the Ca2+ store in the endothelium may then trigger influx of extracellular Ca2+, contributing to an increase in free Ca2+ in the endothelial cells, which activates NO synthase and NO formation.
Asunto(s)
Antiarrítmicos/farmacología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Indoles/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/biosíntesis , Aminoácido Oxidorreductasas/efectos de los fármacos , Aminoácido Oxidorreductasas/metabolismo , Animales , Aorta Torácica/fisiología , Arginina/análogos & derivados , Arginina/farmacología , Calcio/metabolismo , Calcio/farmacología , Calmodulina/antagonistas & inhibidores , GMP Cíclico/biosíntesis , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Masculino , Músculo Liso Vascular/fisiología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa , Nitroarginina , Ratas , Ratas Wistar , Estimulación QuímicaRESUMEN
The contribution of endotoxin to the L-arginine-induced relaxation of the endothelium-denuded rat thoracic aorta, which appears to be mediated by nitric oxide synthase in the vascular smooth muscle, was investigated. Special attention was paid to the time course of the phenomenon and its dependence on the concentration of endotoxin. In the absence of endotoxin, L-arginine induced scarcely any relaxation of the arteries. Treatment of the arteries with endotoxin initiated relaxation in response to 10 microM L-arginine with lag periods of 2-4 hours. The degree of relaxation increased on repeated applications of L-arginine, to reach a consistent level after several hours. Increase in the concentration of endotoxin shortened the lag period, enhanced the degree of relaxation and lowered the threshold concentration of L-arginine required to relax the arteries. In endotoxin-primed arteries, L-arginine, at concentrations necessary to induce relaxation, stimulated the cyclic GMP production. Prophylactic application of actinomycin D or dexamethasone, which inhibits the induction of nitric oxide synthase, prevented the induction by endotoxin of the L-arginine-induced relaxation and cyclic GMP formation. Polymyxin B, which inhibits the action of endotoxin, also prevented the development of the endotoxin-sensitized relaxation and the cyclic GMP formation induced by L-arginine. When the Krebs solution was prepared using deionized water, the amount of endotoxin in the reservoir was above the level required to initiate the L-arginine-induced relaxation and cyclic GMP formation. These results suggest that endotoxin triggered the time-dependent development of the L-arginine-induced relaxation by expressing nitric oxide synthase in the vascular smooth muscle.
Asunto(s)
Arginina/farmacología , Endotoxinas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/fisiología , Salmonella typhi , Aminoácido Oxidorreductasas/biosíntesis , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , GMP Cíclico/metabolismo , Dactinomicina/farmacología , Inducción Enzimática/efectos de los fármacos , Glucocorticoides/farmacología , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintasa , Polimixina B/farmacología , Ratas , Ratas WistarRESUMEN
Thapsigargin induced endothelium-dependent relaxation and cGMP production in rat thoracic aorta, and these effects were inhibited by nitric oxide (NO) pathway inhibitors, a calmodulin inhibitor and removal of Ca2+, suggesting that NO is involved in the thapsigargin-induced relaxation. Thapsigargin may deplete Ca2+ stores in the endothelial cells by inhibiting the Ca(2+)-ATPase, a Ca2+ pump, which in turn triggers influx of extracellular Ca2+, leading to activation of constitutive NO synthase and resultant NO generation. The NO thus formed may activate soluble guanylate cyclase to produce cGMP in the vascular smooth muscle.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Relajación Muscular/efectos de los fármacos , Óxido Nítrico/biosíntesis , Terpenos/farmacología , Aminoquinolinas/farmacología , Animales , Aorta Torácica/metabolismo , Arginina/análogos & derivados , Arginina/farmacología , Calcio/fisiología , GMP Cíclico/biosíntesis , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Nitroarginina , Ratas , Ratas Wistar , SRS-A/antagonistas & inhibidores , TapsigarginaRESUMEN
1. The effects of 12-O-tetradecanoyl 4 beta-phorbol 13-acetate (beta-TPA) on the inositol 1,4,5-trisphosphate (IP3) production, Ca2+ release from the intracellular Ca2+ stores and sensitization of contractile apparatus, induced by prostaglandin F2 alpha (PGF2 alpha) and U46619, a thromboxane A2-mimetic, were studied, using fura-2-loaded and -unloaded rat thoracic aortic strips. 2. Both eicosanoids had characteristic patterns of responses in Ca(2+)-free, 2 mM EGTA-containing solution (Ca(2+)-free solution). They induced transient increases in intracellular Ca2+ concentration ([Ca2+]i) without corresponding transient contraction, but produced delayed, sustained contraction, where [Ca2+]i was returned to the basal level. 3. Treatment with beta-TPA for 60 min reduced the eicosanoids-induced IP3 production, suggesting that the treatment inhibits PIP2 breakdown. 4. The treatment also attenuated [Ca2+]i transient induced by the eicosanoids, but not by caffeine (an IP3-independent releaser of stored Ca2+), in fura-2-loaded preparations incubated in Ca(2+)-free solution. 5. In contrast in the presence of beta-TPA, the sustained contractions evoked by the eicosanoids in Ca(2+)-free solution were potentiated, suggesting that the sites of actions of beta-TPA and the eicosanoids may differ from each other. 6. PGF2 alpha and U46619 utilize different and parallel signal transduction pathways to release Ca2+ by IP3 produced by PIP2 breakdown (beta-TPA-sensitive), and to increase the sensitivity of contractile apparatus, in which protein kinase C may not be involved (beta-TPA-insensitive).
Asunto(s)
Calcio/metabolismo , Eicosanoides/farmacología , Músculo Liso Vascular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animales , Aorta Torácica/efectos de los fármacos , Calcio/fisiología , Medios de Cultivo , Dinoprost/farmacología , Flurbiprofeno/farmacología , Fura-2 , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/biosíntesis , Masculino , Contracción Muscular/efectos de los fármacos , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Proteína Quinasa C/farmacología , Quinacrina/farmacología , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol/farmacología , Vasoconstrictores/farmacologíaRESUMEN
1. Endothelin-3 (ET-3) at concentrations below those which caused contraction (30 nM) elicited endothelium-dependent relaxation followed by rebound contraction in rat isolated thoracic aorta. 2. Endothelin-1 also relaxed the rat aorta with a similar potency. 3. The nitric oxide (NO) synthase inhibitor, NG-nitro L-arginine, the radical scavenger, haemoglobin and the soluble guanylate cyclase inhibitor, methylene blue, each inhibited the ET-3-induced relaxation. 4. The calmodulin inhibitor, calmidazolium, considerably attenuated the relaxation caused by ET-3 without affecting that to nitroprusside. 5. Concentrations of ET-3 that were necessary to induce the relaxation also caused concentration-dependent elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels. 6. NG-nitro L-arginine, haemoglobin, methylene blue, calmidazolium and removal of the endothelium completely abolished ET-3-stimulated cyclic GMP production. 7. These results suggest that ET-3 triggers NO formation possibly via ETB receptors on the endothelium to activate soluble guanylate cyclase, which in turn stimulates cyclic GMP production and smooth muscle relaxation. The enzyme contributing to the NO formation may be of the calcium/calmodulin-dependent, constitutive type.
Asunto(s)
Endotelinas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Acetilcolina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Arginina/análogos & derivados , Arginina/farmacología , Calmodulina/antagonistas & inhibidores , GMP Cíclico/biosíntesis , Hemoglobinas/farmacología , Imidazoles/farmacología , Técnicas In Vitro , Masculino , Azul de Metileno/farmacología , Relajación Muscular/efectos de los fármacos , Nitroarginina , Ratas , Ratas WistarRESUMEN
1. Characteristics of L-arginine-induced non-endothelial nitric oxide (NO) formation in rat isolated thoracic aorta were investigated. 2. Relaxation to L-arginine in arterial rings devoid of endothelium developed about 2 h after the first challenge with L-arginine and reached a maximum after a further 4 h of incubation. 3. After the arteries had relaxed in response to L-arginine, guanosine 3':5'-cyclic monophosphate (cyclic GMP) production was stimulated. In fresh arteries that had not yet relaxed in response to L-arginine, L-arginine failed to elevate cyclic GMP levels. 4. Glucocorticoids, actinomycin D and polymyxin B prevented the development of L-arginine-induced relaxation and L-arginine-stimulated increase in cyclic GMP formation. 5. Once L-arginine-induced relaxation developed, these agents no longer suppressed the relaxation and increase in cyclic GMP formation to L-arginine. 6. From these results, it is suggested that in the isolated thoracic aorta of the rat, endotoxin in the medium triggers induction of a non-endothelial NO synthase during prolonged incubation, which accelerates production of NO from added L-arginine to cause relaxation of the arteries via cyclic GMP. Glucocorticoids and protein synthesis inhibitors may prevent induction of NO synthase. It is suggested that the NO synthase mediating the production of muscle-derived NO from L-arginine is an inducible type.
Asunto(s)
Aminoácido Oxidorreductasas/biosíntesis , Aorta Torácica/efectos de los fármacos , Arginina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Animales , Aorta Torácica/fisiología , GMP Cíclico/metabolismo , Dactinomicina/farmacología , Dexametasona/farmacología , Inducción Enzimática , Glucocorticoides/farmacología , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Óxido Nítrico Sintasa , Fenilefrina/farmacología , Biosíntesis de Proteínas , Ratas , Ratas WistarRESUMEN
1. The mechanism of the vasorelaxant effect of platelet activating factor (PAF) on rat thoracic aorta and the effect of aging on the PAF-induced relaxation were investigated. 2. PAF at concentrations causing relaxation induced marked increases in guanosine 3':5'-cyclic monophosphate (cyclic GMP) production, but did not induce an increase in adenosine 3':5'-cyclic monophosphate (cyclic AMP). 3. Removal of the endothelium by mechanical rubbing, and treatment with the PAF antagonists CV-3988, CV-6209 and FR-900452, the nitric oxide biosynthesis inhibitor, NG-nitro L-arginine, the radical scavenger, haemoglobin, and the soluble guanylate cyclase inhibitor, methylene blue, inhibited PAF-induced relaxation and abolished or attenuated PAF-stimulated cyclic GMP production. 4. The relaxation was greatest in arteries from rats aged 4 weeks. With an increase in age, the response of the arteries to PAF was attenuated. 5. Endothelium-dependent cyclic GMP production also decreased with increase in age of the rats. 6. These results suggest that PAF stimulates production of nitric oxide from L-arginine by acting on the PAF receptors in the endothelium, which in turn stimulates soluble guanylate cyclase in the smooth muscle cells, and so increases production of cyclic GMP, thus relaxing the arteries. Age-associated decrease in PAF-induced relaxation may result from a reduction of cyclic GMP formation.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Factor de Activación Plaquetaria/farmacología , Envejecimiento/fisiología , Animales , Aorta Torácica , Arginina/análogos & derivados , Arginina/farmacología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Endotelio Vascular/fisiología , Guanilato Ciclasa/metabolismo , Hemoglobinas/farmacología , Masculino , Azul de Metileno/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/fisiología , NG-Nitroarginina Metil Éster , Factor de Activación Plaquetaria/antagonistas & inhibidores , Compuestos de Piridinio/farmacología , Ratas , Ratas WistarRESUMEN
Contractile mechanisms by carbachol and ATP were compared in the detrusor strips of rabbit bladder. To exclude modulation of the agonists-induced responses by intramurally synthesized prostaglandins, all the experiments were done in the presence of the potent cyclooxygenase inhibitor flurbiprofen (1 microM). The concentration-response curves for carbachol and ATP were shifted to the right by 6- 10-fold by verapamil (10 microM), which abolished the K-induced contraction of the atropinized detrusor completely. A similar curve for carbachol was obtained in the absence of extracellular Ca [(Ca)o], but the contraction by ATP below 1 mM was more reduced by (Ca)o-depletion than by verapamil. Under Ca-free conditions, repeated applications of ATP resulted in no response, but those of carbachol induced reproducible contractions. These results suggest that carbachol and ATP induces Ca-influx through L-type Ca channels and releases Ca from the Ca stores. However, while carbachol might increase the sensitivity of contractile machinery to Ca on the one hand, ATP would open additional, verapamil-insensitive Ca channels.
Asunto(s)
Adenosina Trifosfato/farmacología , Carbacol/farmacología , Músculo Liso/efectos de los fármacos , Animales , Calcio/fisiología , Inhibidores de la Ciclooxigenasa/farmacología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Potasio/farmacología , Conejos , Receptores Purinérgicos/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , Verapamilo/farmacologíaRESUMEN
We investigated the effect of aging on atrial natriuretic peptide (ANP)-induced relaxation and cyclic GMP (cGMP) formation in the rat thoracic aorta. In the aorta from young rats (4 weeks old), removal of the endothelium, and treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), the radical scavenger, hemoglobin (Hb), and the soluble guanylate cyclase inhibitor, methylene blue (MB), attenuated ANP-induced relaxation and considerably reduced ANP-stimulated cGMP formation. With increasing age of the rats, the ANP-induced relaxation and cGMP formation in endothelium-intact aorta decreased, and Hb, L-NAME and MB no longer inhibited the ANP-induced effects, irrespective of whether the endothelium was present or absent. In the arteries without endothelium, the age-associated reduction in ANP-induced relaxation was less than in arteries with endothelium. Aging also decreased the relaxation induced by the soluble guanylate cyclase activator, nitroprusside. Potentiation due to the cGMP-phosphodiesterase (cGMP-PDE) inhibitor, M&B 22948, of the ANP-induced relaxation was greater in aortas from old rats than in those from young rats, suggesting that the degradation of cGMP may be accelerated in old rats. These results suggest that the relaxant action of ANP on the thoracic aorta from young rats is in part modulated by endothelium-derived relaxing factor (EDRF/nitric oxide), which in turn activates soluble guanylate cyclase, thus elevating the cGMP level. Aging may decrease the ANP-induced relaxation and ANP-stimulated increase in cGMP level by decreasing the ability of endothelial cells to produce EDRF, by decreasing guanylate cyclase activity, and by enhancing cGMP-PDE activity.
Asunto(s)
Envejecimiento/fisiología , Factor Natriurético Atrial/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiología , Arginina/análogos & derivados , Arginina/farmacología , GMP Cíclico/biosíntesis , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Guanilato Ciclasa/metabolismo , Hemoglobinas/farmacología , Técnicas In Vitro , Masculino , Azul de Metileno/farmacología , Relajación Muscular/fisiología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , NG-Nitroarginina Metil Éster , Purinonas/farmacología , Ratas , Ratas EndogámicasRESUMEN
1. The relaxation of rings of rat thoracic aorta induced by L-arginine and its derivatives was investigated. 2. L-Arginine (0.3-100 microM), but not D-arginine, induced relaxation of the arteries, which was detectable after 2 h and maximal after 4-6 h on its repeated application; it was endothelium-independent. 3. L-Arginine methyl ester, N alpha-benzoyl L-arginine and L-homo-arginine had essentially similar effects to those of L-arginine. 4. NG-nitro L-arginine methyl ester (L-NAME, 3 microM), NG-nitro L-arginine (L-NNA, 1 microM) and NG-monomethyl L-arginine (L-NMMA, 10 microM), inhibitors of nitric oxide (NO) formation from L-arginine, inhibited or reversed the L-arginine-induced relaxation, irrespective of the presence or absence of the endothelium. In contrast, NG-nitro D-arginine was without effect. 5. Haemoglobin (Hb, 10 nM) and methylene blue (MB, 0.3 microM) inhibited or reversed the L-arginine-induced relaxation. 6. L-Arginine (1-100 microM), but not D-arginine, increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in the tissues that relaxed in response to L-arginine. This effect of L-arginine was suppressed by Hb (3 microM), MB (1 microM) and L-NAME (100 microM). Removal of the endothelium did not significantly alter the L-arginine-induced cyclic GMP production. 7. These results suggest that L-arginine itself caused a slowly developing relaxation of rat aorta, possibly via formation of NO by an endothelium-independent mechanism.
Asunto(s)
Arginina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Arginina/análogos & derivados , AMP Cíclico/metabolismo , GMP Cíclico/biosíntesis , GMP Cíclico/metabolismo , Endotelio Vascular/fisiología , Hemoglobinas/farmacología , Técnicas In Vitro , Masculino , Azul de Metileno/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster , Nitroarginina , Ratas , Ratas EndogámicasRESUMEN
From functional studies with propylbenzilylcholine mustard (PrBCM), we reported that there coexist PrBCM-sensitive and PrBCM-resistant muscarinic cholinoceptor mechanisms in guinea pig taenia caecum. We investigated the interrelationship between these two cholinoceptor mechanisms using an in vitro receptor binding assay with [3H]quinuclidinyl benzilate (QNB) and [3H]PrBCM. Pretreatment of the muscle strips with 300 nM PrBCM (in vivo alkylation) for 10-50 min resulted in progressive decreases of the number of the maximum [3H]QNB binding sites. However, a prolongation of the period of in vivo alkylation up to 90 min was accompanied with no further loss in the binding sites. Under these conditions, there is no significant change in the affinity of [3H]QNB for the binding sites. The concentration of carbachol required to displace 50% of the bound [3H]QNB was larger in membranes obtained from the tissues that had been alkylated in vivo with PrBCM for 50 min than that from control strips, but was not altered when the pretreatment with the drug was carried out after homogenization (in vitro alkylation). When GTP was added during in vitro alkylation, the affinity of carbachol was lower than that in control membranes, as observed when in vivo alkylation was carried out. In the presence of guanine nucleotide, PrBCM thus appears to recognize two distinct populations or states of muscarinic receptors.
Asunto(s)
Guanosina Trifosfato/farmacología , Músculo Liso/efectos de los fármacos , Mostaza de Propilbencililcolina/farmacología , Receptores Muscarínicos/efectos de los fármacos , Alquilación , Animales , Carbacol/farmacología , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Cobayas , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Cinética , Membranas/efectos de los fármacos , Proteínas/metabolismo , Quinuclidinil BencilatoRESUMEN
1. In rat tail artery, endothelin-1(ET-1) caused an increase in the cytosolic free Ca2+ level ([Ca2+]i) followed by a relatively sustained but not steady-state contraction in Ca2+ containing solution. 2. In the early phase of the contraction, the rate of increase in [Ca2+]i, was much faster than that in muscle tension. However, after the increases in [Ca2+]i and muscle tension reached at their peaks, there was a good correlation between the changes of the two parameters. 3. ET-1 could not induce an apparent contractile response in Ca2(+)-free medium, notwithstanding it evoked a [Ca2+]i transient in this medium. 4. The results indicate that ET-1 induce a contraction of rat tail artery which is almost fully dependent on the [Ca2+]i changes, and may inhibit the Ca2(+)-sensitivity of the contractile filaments in the early phase of the contraction.
Asunto(s)
Calcio/fisiología , Endotelinas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Animales , Arterias/efectos de los fármacos , Cafeína/farmacología , Calcio/metabolismo , Femenino , Fura-2/farmacología , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Endogámicas , Flujo Sanguíneo Regional/efectos de los fármacos , Cola (estructura animal)/irrigación sanguíneaRESUMEN
1. The effects of ryanodine on changes in cytoplasmic Ca2+ level ([Ca2+]i) and muscle tension induced by maximum concentrations of phenylephrine (Phe; 1 microM), prostaglandin F2 alpha (PGF2 alpha, 10 microM), caffeine (Caf, 30 mM) and isoprenaline (Iso, 1 microM) were examined in rat aortic strips using fura-2. 2. In normal media, Phe and PGF2 alpha produced a phasic contraction, followed by a tonic one. Caf elicited only a transient contraction. When the preparation was treated with 10 microM ryanodine, an increase in [Ca2+]i was induced accompanied by a nicardipine (1 microM)-resistant contraction which was [Ca2+]o-dependent. 3. In Ca2(+)-free solution, the three stimulants elicited transient increases in [Ca2+]i. Transient contractions to Phe and Caf were accompanied by changes in [Ca2+]i. The transient increase in [Ca2+]i induced by PGF2 alpha was not accompanied by a corresponding contraction. 4. Sustained contractions were induced by Phe and PGF2 alpha in the absence of external Ca2+, while the increase in [Ca2+]i was reduced. A larger maximum contraction was induced by PGF alpha than by Phe. 5. Ryanodine abolished both the Caf- and Phe-induced [Ca2+]i transient increases and the corresponding contractions, but had no substantial effect on the PGF2 alpha-induced [Ca2+]i transient increase. Ryanodine had no influence on the sustained contractions induced by Phe and PGF2 alpha. 6. Iso relaxed both sustained contractions almost completely, without any detectable change in [Ca2+]i. Treatment of the preparation with ryanodine had no effect on the concentration-response curves for Iso in relaxing the 0.1 microM Phe- or 40 mM K(+)-induced precontraction. 7. It is suggested that Phe and Caf mobilize Ca2 + from a ryanodine-sensitive Ca2 + store and that PGF2 alpha. releases Ca2+ from a ryanodine-insensitive Ca2+ store. The former contributes to the transient contraction through a Ca2'-dependent process, while the latter seems not to be directly associated with the contraction. The sustained contraction under Ca2+-free conditions might involve a Ca2 '-independent process or a change in the sensitivity of the contractile filaments to Ca2 + 8. In addition to lowering cytoplasmic Ca2+ concentration, it is suggested that Iso counteracts the apparently Ca2 +-independent process. The ryanodine-sensitive Ca2+ store plays no substantial role in active relaxation by Iso, although it does play a major role in the maintenance of cytoplasmic Ca2+ in a quiescent muscle.
Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Rianodina/farmacología , Animales , Cafeína/farmacología , Citoplasma/fisiología , Dinoprost/farmacología , Ácido Egtácico/farmacología , Fura-2 , Isoproterenol/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Fenilefrina/farmacología , Ratas , Ratas Endogámicas , Vasodilatación/efectos de los fármacosRESUMEN
The modes by which endothelin-1 (ET) induces Ca2(+)-influx and the relative functional importance of the different sources of Ca2+ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca2+ level ([Ca2+]i) followed by a tonic contraction in Ca2(+)-containing solution, and produced a transient elevation of [Ca2+]i followed by a small sustained contraction in Ca2(+)-free medium. ET also stimulated 45Ca influx into La3(+)-inaccessible fraction significantly. With the same change of [Ca2+]i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca2+]i elevation were not significantly inhibited by 0.1-0.3 microM nicardipine which nearly abolished the contraction and [Ca2+]i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca2+]i and muscle tension, and vice versa. In Ca2(+)-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca2+]i transient induced by ET. Further, the ET-induced sustained contraction under Ca2(+)-free conditions began to develop after the [Ca2+]i level returned to the baseline. Thus, it seems that the Ca2+ released from the ryanodine-sensitive and -insensitive Ca2+ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca2(+)-influx through Ca2+ channel(s) other than voltage-dependent Ca2+ channels in character, and by increasing the sensitivity of the contractile filaments to Ca2+ or activating them Ca2(+)-independently.