RESUMEN
In recent years, nanocarriers have played an ever-increasing role in clinical and biomedical applications owing to their unique physicochemical properties and surface functionalities. Lately, much effort has been directed towards the development of smart, stimuli-responsive nanocarriers that are capable of releasing their cargos in response to specific stimuli. These intelligent-responsive nanocarriers can be further surface-functionalized so as to achieve active tumor targeting in a sequential manner, which can be simply modulated by the stimuli. By applying this methodological approach, these intelligent-responsive nanocarriers can be directed to different target-specific organs, tissues, or cells and exhibit on-demand controlled drug release that may enhance therapeutic effectiveness and reduce systemic toxicity. Light, an external stimulus, is one of the most promising triggers for use in nanomedicine to stimulate on-demand drug release from nanocarriers. Light-triggered drug release can be achieved through light irradiation at different wavelengths, either in the UV, visible, or even NIR region, depending on the photophysical properties of the photo-responsive molecule embedded in the nanocarrier system, the structural characteristics, and the material composition of the nanocarrier system. In this review, we highlighted the emerging functional role of light in nanocarriers, with an emphasis on light-responsive liposomes and dual-targeted stimuli-responsive liposomes. Moreover, we provided the most up-to-date photo-triggered targeting strategies and mechanisms of light-triggered drug release from liposomes and NIR-responsive nanocarriers. Lastly, we addressed the current challenges, advances, and future perspectives for the deployment of light-responsive liposomes in targeted drug delivery and therapy.
Asunto(s)
Nanopartículas , Neoplasias , Humanos , Liposomas/uso terapéutico , Portadores de Fármacos/química , Nanopartículas/química , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológicoRESUMEN
AIMS: Cigarette smoke often induces pulmonary and systemic inflammation. In animal models, mesenchymal stem cells (MSC) tend to ameliorate these effects. We aimed to explore the local and systemic expression of cytokines in guinea pigs chronically exposed to cigarette smoke, and their modifications by MSC. MAIN METHODS: Concentrations of IL-1ß, IL-6, IL-8, IL-12, TNF-α, INF-É£, TSG-6, MMP-9, TIMP-1, and/or TIMP-2 in serum and bronchoalveolar lavage (BALF) from animals exposed to tobacco smoke (20 cigarettes/day, 5 days/week for 10 weeks) were determined, and mRNA expression of some of them was measured in lung tissue. Intratracheal instillation of allogeneic bone marrow MSC (5x106 cells in 1 ml) was done at week 2. KEY FINDINGS: After cigarette smoke, IL-6 and IFN-γ increased in serum and BALF, while IL-1ß and IL-12 decreased in serum, and TSG-6 and TIMP-2 increased in BALF. IL-1ß had a paradoxical increase in BALF. MSC had an almost null effect in unexposed animals. The intratracheal administration of MSC in guinea pigs exposed to cigarette smoke was associated with a statistically significant decrease of IL-12 and TSG-6 in serum, as well as a decrease of IL-1ß and IFN-γ and an increase in TIMP-1 in BALF. Concerning mRNA expression in lung tissue, cigarette smoke did not modify the relative amount of the studied transcripts, but even so, MSC decreased the IL-12 mRNA and increased the TIMP-1 mRNA. SIGNIFICANCE: A single intratracheal instillation of MSC reduces the pulmonary and systemic proinflammatory pattern induced by chronic exposure to cigarette smoke in guinea pigs. TRIAL REGISTRATION: Not applicable.
Asunto(s)
Fumar Cigarrillos , Células Madre Mesenquimatosas , Cobayas , Animales , Citocinas/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2 , Interleucina-6/farmacología , Fumar Cigarrillos/efectos adversos , Pulmón/metabolismo , Interleucina-12/farmacología , ARN Mensajero , Células Madre Mesenquimatosas/metabolismo , Líquido del Lavado BronquioalveolarRESUMEN
Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm after nebulization with ATP. Because the main mechanism for controlling the noxious effects of extracellular ATP is its enzymatic hydrolysis, we hypothesized that allergic sensitization is accompanied by a decreased functioning of such hydrolysis. In the present study, peripheral blood leukocytes from sensitized and non-sensitized guinea pigs were used for determining the extracellular metabolism (as assessed by inorganic phosphate production) of ATP, ADP, AMP, or adenosine, and for detecting possible changes in the expression (qPCR and Western blot) of major ectonucleotidases (NTPDase1, NTPDase3, and NPP1) and purinoceptors (P2X1, P2X7, P2Y4, and P2Y6). Contrary to our hypothesis, we found that leukocytes from allergic animals produced higher amounts of inorganic phosphate after stimulation with ATP and ADP, as compared with leukocytes from non-sensitized animals. Although at first glance, this result suggested that sensitization caused higher efficiency of ectonucleotidases, their mRNA and protein expressions were unaffected. On the other hand, after sensitization, we found a significant increase in the protein expression of P2X7 and P2Y4, two purinoceptors known to be responsible for ATP release after activation. We concluded that allergic sensitization increased the amount of ATP hydrolyzed by ectonucleotidases, the latter probably not due to the enhanced efficiency of its enzymatic breakdown, but rather due to an increased release of endogenous ATP or other nucleotides, partly mediated by enhanced expression or P2X7 and P2Y4 receptors.
Asunto(s)
Adenosina Trifosfato/metabolismo , Hipersensibilidad/metabolismo , Leucocitos/metabolismo , Animales , Cobayas , Hidrólisis , Masculino , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMEN
UNLABELLED: Membrane depolarization of airway smooth muscle (ASM) opens L-type voltage dependent Ca(2+) channels (L-VDCC) allowing Ca(2+) entrance to produce contraction. In Ca(2+) free conditions Na(+) permeates through L-VDCC in excitable and non-excitable cells and this phenomenon is annulled at µM Ca(2+) concentrations. Membrane depolarization also induces activation of Gq proteins and sarcoplasmic reticulum Ca(2+) release. In bovine ASM, KCl induced a transient contraction sensitive to nifedipine in Ca(2+)free medium, indicating an additional mechanism to the SR-Ca(2+) release. It is possible that Na(+) could permeate through L-VDCC in bovine ASM. KCl induced a transient contraction in Ca(2+) free medium with a fast intracellular Ca(2+) increment, reduced by TMB-8. This contraction was abolished by caffeine and CPA, diminished with nifedipine and augmented by Bay K8644. Increasing extracellular Na(+) concentration in tracheal myocytes, proportionally augmented the SBFI fluorescence ratio, suggesting an increment in the intracellular Na(+) concentration ([Na(+)]i). 50mM Na(+) with and without Ca(2+) induced a [Na(+)]i increment, enhanced by Bay K8644 and inhibited with D-600. In Ca(2+) free medium, KCl increased [Na(+)]i. Ba(2+) currents corresponding to L-VDCC were observed in myocytes and Na(+) permeated in the presence and absence of Ca(2+). SBFI-loaded myocytes in Na(+) and Ca(2+) containing Krebs stimulated with carbachol showed a Na(+) increment with a plateau. D-600 and 2-APB almost abolished the carbachol-induced Na(+) increment. RT-PCR demonstrated that CaV1.2 is the only L-VDCC subunit present in ASM. CONCLUSION: under physiological conditions, Na(+) permeates through L-VDCC in bovine ASM, probably contributing to sustain membrane depolarization during agonist stimulation.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Músculo Liso/metabolismo , Sodio/metabolismo , Tráquea/metabolismo , Animales , Canales de Calcio Tipo L/genética , Bovinos , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Músculo Liso/citología , Permeabilidad , Retículo Sarcoplasmático/metabolismoRESUMEN
The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with ß1 integrins (α1ß1 and α2ß1) in guinea pig. The putative role of ß1 integrin subunit alterations due to shedding in airway smooth muscle (ASM) in an allergic asthma model was evaluated. Guinea pigs were sensitized and challenged with antigen. Antigenic challenges induced bronchoobstruction and hyperresponsiveness at the third antigenic challenge. Immunohistochemistry and immunoelectronmicroscopy studies showed that the cytosolic and extracellular domains of the ß1 integrin subunit shared the same distribution in airway structures in both groups. Various polypeptides with similar molecular weights were detected with both the cytosolic and extracellular ß1 integrin subunit antibodies in isolated airway myocytes and the connective tissue that surrounds the ASM bundle. Flow cytometry and Western blot studies showed that the expression of cytosolic and extracellular ß1 integrin subunit domains in ASM was similar between groups. An increment of ITGB1 mRNA in ASM was observed in the asthma model group. RACE-PCR of ITGB1 in ASM did not show splicing variants. The expression levels of integrin-linked kinase (ILK) and paxillin diminished in the asthma model, but not talin. The levels of phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr(696) increased in asthma model. Our work suggests that ß1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, ß1 integrin cytodomain assembly proteins in focal cell adhesions in which ILK and paxillin are involved are altered in asthma model. J. Cell. Biochem. 117: 2385-2396, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Asma/inmunología , Modelos Animales de Enfermedad , Adhesiones Focales/metabolismo , Integrina beta1/metabolismo , Músculo Liso/inmunología , Sistema Respiratorio/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Animales , Asma/metabolismo , Asma/patología , Western Blotting , Células Cultivadas , Cobayas , Masculino , Músculo Liso/metabolismo , Músculo Liso/patología , Paxillin/antagonistas & inhibidores , Paxillin/genética , Paxillin/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sistema Respiratorio/metabolismo , Sistema Respiratorio/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Talina/antagonistas & inhibidores , Talina/genética , Talina/metabolismoRESUMEN
AIMS: Almost every eukaryotic cell releases ATP under certain conditions. The idea that ATP induces the release of ATP has been scantly investigated. METHODS: We explored this possibility by assessing the rate of exogenous ATP breakdown (measured by phosphates production) by human peripheral blood leukocytes. The role of P2Y and P2X receptors was evaluated pharmacologically, by patch clamp, or by flow cytometry. KEY FINDINGS: In mononuclear and/or polymorphonuclear cells, ATP increased phosphates formation in a time- and concentration-dependent manner. Uncoupling of P2Y receptors with N-ethylmaleimide and antagonism of P2Y and P2X receptors through suramin reduced phosphate formation after 500µM ATP, suggesting that part of the phosphate production was due to activation of P2 receptors, with subsequent release of ATP or other nucleotides. Similar results were obtained with UTP and ATPγS. Gadolinium (connexins inhibitor) also significantly reduced the ATP-induced phosphate production. Blockade of P2X receptors with SKF 96365 or NF023 did not modify the phosphate production. In monocytes, 500µM ATP induced inward currents suggestive of P2X1 activation, but higher concentrations (1-5mM) induced inward currents suggestive of P2X7 activation. We discarded a role of adenosine in the ATP-evoked nucleotides release. Flow cytometry identified that almost all mononuclear and polymorphonuclear cells expressed P2Y1,2,4,6,11 receptors. SIGNIFICANCE: 500µM ATP induced the release of ATP or other nucleotides through activation of P2Y2,4,6,11 receptors in human leukocytes, and probably via P2X receptors at higher concentrations. This ATP-induced nucleotides release constitutes a potential mechanism leading to amplification of ATP signaling.
Asunto(s)
Adenosina Trifosfato/metabolismo , Leucocitos Mononucleares/metabolismo , Nucleótidos/metabolismo , Receptores Purinérgicos P2/metabolismo , Humanos , Transducción de SeñalRESUMEN
There is mounting evidence that 5-hydroxytryptamine (5-HT) plays a role in asthma. However, scarce information exists about the pulmonary expression of 5-HT receptors and its modification after allergic sensitization. In the present work, we explored the expression of 5-HT1A, 5-HT2A, 5-HT3, 5-HT4, 5-ht5a, 5-HT6, and 5-HT7 receptors in lungs from control and sensitized guinea pigs through qPCR and Western blot. In control animals, mRNA from all receptors was detectable in lung homogenates, especially from 5-HT2A and 5-HT4 receptors. Sensitized animals had decreased mRNA expression of 5-HT2A and 5-HT4 receptors and increased that of 5-HT7 receptor. In contrast, they had increased protein expression of 5-HT2A receptor in bronchial epithelium and of 5-HT4 receptor in lung parenchyma. The degree of airway response to the allergic challenge was inversely correlated with mRNA expression of the 5-HT1A receptor. In summary, our results showed that major 5-HT receptor subtypes are constitutively expressed in the guinea pig lung, and that allergic sensitization modifies the expression of 5-HT2A, 5-HT4, and 5-HT7 receptors.
Asunto(s)
Asma/metabolismo , Hipersensibilidad/metabolismo , Inmunización , Receptores de Serotonina/metabolismo , Animales , Modelos Animales de Enfermedad , Cobayas , Masculino , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Serotonina 5-HT4/metabolismoRESUMEN
Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.
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Adenosina Trifosfato/metabolismo , Carbacol/farmacología , Ciclooxigenasa 2/metabolismo , Histamina/farmacología , Serotonina/farmacología , Tráquea/efectos de los fármacos , Animales , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa 2/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Cobayas , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , ARN Mensajero/genética , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Tromboxano A2/genética , Tromboxano A2/metabolismo , Tráquea/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology and uncertain pathogenic mechanisms. Recent studies indicate that the pathogenesis of the disease may involve the abnormal expression of certain developmental pathways. Here we evaluated the expression of Sonic Hedgehog (SHH), Patched-1, Smoothened, and transcription factors glioma-associated oncogene homolog (GLI)1 and GLI2 by RT-PCR, as well as their localization in IPF and normal lungs by immunohistochemistry. The effects of SHH on fibroblast proliferation, migration, collagen and fibronectin production, and apoptosis were analyzed by WST-1, Boyden chamber chemotaxis, RT-PCR, Sircol, and annexin V-propidium iodide binding assays, respectively. Our results showed that all the main components of the Sonic signaling pathway were overexpressed in IPF lungs. With the exception of Smoothened, they were also upregulated in IPF fibroblasts. SHH and GLI2 localized to epithelial cells, whereas Patched-1, Smoothened, and GLI1 were observed mainly in fibroblasts and inflammatory cells. No staining was detected in normal lungs. Recombinant SHH increased fibroblast proliferation (P < 0.05), collagen synthesis, (2.5 ± 0.2 vs. 4.5 ± 1.0 µg of collagen/ml; P < 0.05), fibronectin expression (2-3-fold over control), and migration (190.3 ± 12.4% over control, P < 0.05). No effect was observed on α-smooth muscle actin expression. SHH protected lung fibroblasts from TNF-α/IFN-γ/Fas-induced apoptosis (14.5 ± 3.2% vs. 37.3 ± 7.2%, P < 0.0001). This protection was accompanied by modifications in several apoptosis-related proteins, including increased expression of X-linked inhibitor of apoptosis. These findings indicate that the SHH pathway is activated in IPF lungs and that SHH may contribute to IPF pathogenesis by increasing the proliferation, migration, extracellular matrix production, and survival of fibroblasts.
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Proteínas Hedgehog/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Apoptosis , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiología , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiología , Humanos , Fibrosis Pulmonar Idiopática/patología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Pulmón/metabolismo , Pulmón/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Receptor Smoothened , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de ZincRESUMEN
In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,ß-methylene-ATP was inactive from 1 to 100 µM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.
Asunto(s)
Músculo Liso/fisiología , Canales de Potasio/fisiología , Receptores Purinérgicos P2Y/fisiología , 4-Aminopiridina/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Adenosina Trifosfato , Animales , Etilmaleimida/farmacología , Cobayas , Masculino , Relajación Muscular/efectos de los fármacos , Péptidos/farmacología , Prostaglandinas , Suramina/farmacología , Tráquea/fisiología , Triazinas/farmacologíaRESUMEN
In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca(2+)](i). Cytosolic Ca(2+) is extruded to the extracellular space by the Na(+)/Ca(2+) exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCX(REV)), inducing Ca(2+) entry. We investigated whether ATP, via P2X receptors, activates the NCX(REV) and whether the increment in [Ca(2+)](i) is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca(2+)](i) not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca(2+)](i), (a Ca(2+) peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca(2+) peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca(2+)](i) increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCX(REV) and this effect was abolished by SKF 96365 and TNP-ATP (P2X(1) and P2X(3) receptors antagonist). P2X(1) and P2X(3) receptors were corroborated by immunoblotting of ASM. NCX(REV) activation and ATP in the presence of RB2 favor the SR Ca(2+) refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca(2+)](i) through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X(1) and P2X(3) receptors and the NCX(REV) which refills the SR.
Asunto(s)
Adenosina Trifosfato/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Receptores Purinérgicos P2X/fisiología , Receptores Purinérgicos P2Y/fisiología , Retículo Sarcoplasmático/metabolismo , Tráquea/efectos de los fármacos , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Cobayas , Indometacina/farmacología , Masculino , Músculo Liso/efectos de los fármacos , Técnicas de Placa-Clamp , Prostaglandinas/biosíntesis , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Suramina/farmacología , Tráquea/fisiología , Triazinas/farmacologíaRESUMEN
Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H(1)) and 5-hydroxytryptamine (serotonin) receptors (5-HT(2A)) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (approximately 80%), 5-HT (100%), alpha-methyl-5-HT (100%), and KCl (approximately 30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (approximately 85%) and 5-HT (approximately 59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca(2+) that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H1, 5-HT(2A), and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H(1) receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT(2A) receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H(1) and 5-HT(2A) receptors through their Rho/ROCK signaling.
Asunto(s)
Caveolas/fisiología , Colesterol/fisiología , Lípidos de la Membrana/fisiología , Músculo Liso/fisiología , Receptor de Serotonina 5-HT2A/fisiología , Receptores Histamínicos H1/fisiología , Tráquea/fisiología , Quinasas Asociadas a rho/fisiología , Animales , Calcio/metabolismo , Bovinos , Caveolina 1/fisiología , Transducción de Señal , beta-Ciclodextrinas/farmacologíaRESUMEN
AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 microM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 microM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 microM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.
Asunto(s)
Músculo Liso/fisiología , Canales de Potasio Calcio-Activados/fisiología , Sistema Respiratorio/citología , Agonistas del Receptor de Serotonina 5-HT2 , Antagonistas del Receptor de Serotonina 5-HT2 , Serotonina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Antagonistas Adrenérgicos beta/farmacología , Animales , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Cobayas , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Calcio-Activados/agonistas , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/patología , Sistema Respiratorio/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
Androgen vasorelaxing action is a subject of recent interest. We investigated the involvement of l-type voltage-operated Ca(2+) channels (L-VOCCs), K(+) channels, intracellular Ca(2+) concentration ([Ca(2+)]i), and cAMP in the vasorelaxing effect of testosterone and 5beta-dihydrotestosterone (5beta-DHT) on rat thoracic aorta. Isolated aortic rings were used to study the vasorelaxing potency of testosterone and 5beta-DHT on KCl- and noradrenaline-induced contractions. Patch-clamp was used to analyze androgen effects on Ca(2+) inward and K(+) outward currents. The fluorescence technique was used to evaluate [Ca(2+)]i in single myocytes; moreover, simultaneous measurements of [Ca(2+)]i and vascular contraction were evaluated. 5beta-DHT was more potent than testosterone to relax KCl-induced contraction, but they were equipotent to relax noradrenaline contraction. l-type Ca(2+) currents were blocked by nifedipine, both androgens, and an estrogen in a concentration-dependent manner, and the order of potency was: testosterone > nifedipine > 5beta-DHT > 17beta-estradiol. We observed that testosterone has different mechanism of action by the concentration range used: at nm concentrations it was a powerful L-VOCCs antagonist, whereas at mum concentrations it was observed that: 1) its Ca(2+) antagonist property is reverted by increasing the l-type inward Ca(2+) currents (Ca(2+) agonist property); and 2) the [Ca(2+)]i and cAMP production was increased. The total K(+) currents were unaffected by testosterone or 5beta-DHT. The data show that 5beta-DHT-induced vasorelaxation is due to its selective blockade on L-VOCCs (from nm to microm concentrations), but testosterone-induced vasorelaxation involves concentration-dependent additional mechanisms: acting as an L-VOCCs antagonist at low concentrations, and increasing [Ca(2+)]i and cAMP production at high concentrations.
Asunto(s)
Andrógenos/farmacología , Aorta Torácica/efectos de los fármacos , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Dihidrotestosterona/farmacología , Testosterona/farmacología , Vasodilatación/efectos de los fármacos , Animales , Aorta Torácica/fisiología , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Masculino , Potenciales de la Membrana/efectos de los fármacos , Células Musculares , Concentración Osmolar , Potasio/metabolismo , Ratas , Ratas Wistar , Especificidad por SustratoRESUMEN
Airway hyperresponsiveness is a key feature of asthma, but its mechanisms remain poorly understood. Leukotriene D(4) (LTD(4)) is one of the few molecules capable of producing airway hyperresponsiveness. In this study, LTD(4), but not leukotriene C(4) (LTC(4)), produced a leftward displacement of the concentration-response curve to histamine in bovine airway smooth muscle strips. Neither LTC(4) nor LTD(4) modified the concentration-response curve to carbachol. In simultaneous measurements of intracellular Ca(2+) ([Ca(2+)](i)) and contraction, histamine or carbachol produced a transient Ca(2+) peak followed by a plateau, along with a contraction. LTD(4) increased the histamine-induced transient Ca(2+) peak and contraction but did not modify responses to carbachol. Enhanced responses to histamine induced by LTD(4) were not modified by staurosporine or chelerythrine but were abolished by genistein. Western blot showed that carbachol, but not histamine, caused intense phosphorylation of extracellular signal-regulated kinase 1/2 and that LTD(4) significantly enhanced the phosphorylation induced by histamine, but not by carbachol. L-type Ca(2+) channel participation in the hyperresponsiveness to histamine was discarded because LTD(4) did not modify the [Ca(2+)](i) changes induced by KCl. In tracheal myocytes, LTD(4) enhanced the transient Ca(2+) peak induced by histamine (but not by carbachol) and the sarcoplasmic reticulum (SR) Ca(2+) refilling. Genistein abolished this last LTD(4) effect. Partial blockade of the SR-ATPase Ca(2+) pump with cyclopiazonic acid reduced the Ca(2+) transient peak induced by histamine but not by carbachol. These results suggested that LTD(4) induces hyperresponsiveness to histamine through activation of the tyrosine kinase pathway and an increasing SR-ATPase Ca(2+) pump activity. L-type Ca(2+) channels seemed not to be involved in this phenomenon.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Histamina/farmacología , Leucotrieno D4/farmacología , Contracción Muscular , Músculo Liso/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Retículo Sarcoplasmático/enzimología , Tráquea/efectos de los fármacos , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Concentración Osmolar , Fosforilación/efectos de los fármacosRESUMEN
1. Caffeine has been widely used as a pharmacological tool to evaluate Ca(2+) release from the sarcoplasmic reticulum in isolated smooth muscle cells. However, in nervous tissue this drug also causes neurotransmitters release, which might cause additional effects when smooth muscle strips are evaluated. To assess this last possibility, simultaneous measurements of contraction and cytosolic Ca(2+) concentration (using Fura-2/AM) were carried out in bovine airway smooth muscle strips during caffeine stimulation. 2. A first stimulation (S1, n=11) with caffeine (10 mM) induced a biphasic change in cytosolic Ca(2+), which consisted of a transient Ca(2+) peak (254+/-40 nM, X+/-SEM) followed by a plateau (92+/-13 nM), and a transient contraction (204.72+/-31.56 mg tension mg tissue(-1)). A second caffeine stimulation (S2) produced a similar response but these parameters had a different magnitude. The S2/S1 ratios for these parameters were 0.69+/-0.02, 0.83+/-0.06 and 1.01+/-0.03, respectively. Addition of omega-conotoxin GVIA (1 micro M) and tetrodotoxin (3.1 micro M) before S2 significantly diminished these S2/S1 ratios (0.26+/-0.05, 0.26+/-0.09 and 0.64+/-0.11, respectively, n=5, P<0.05), implicating the neurotransmitters release involvement in the response to caffeine. A similar effect (P<0.01) was observed with atropine (1 micro M, n=4), the fragment 4-11 of substance P (SP) (an SP receptor antagonist, 10 micro M, n=5), and with both substances (n=4). 3. We discarded a direct effect of omega-conotoxin GVIA (1 micro M) plus tetrodotoxin (3.1 micro M) or of atropine (1 micro M) plus SP fragment 4-11 on smooth muscle cells because they did not modify caffeine responses in isolated tracheal myocytes. 4. We confirmed by HPLC that caffeine increased the release of acetylcholine (from 0.43+/-0.19 to 2.07+/-0.56 nM mg tissue(-1), P<0.02) in bovine airway smooth muscle strips. Detection of substance P by ELISA was not statistically different after caffeine stimulation (geometric means before and after caffeine, 0.69 vs. 1.97 pg ml(-1) mg tissue(-1), respectively, P=0.053). 5. We concluded that acetylcholine and tachykinins release are involved in the caffeine-induced biphasic changes in cytosolic Ca(2+) concentration.