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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.Concentrations of IL-1β, IL-6, IL-8, IL-12, TNF-α, INF-ɣ, 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 (5x10After cigarette smoke, IL-6 and IFN-γ increased in serum and BALF, while IL-1β and IL-12 decreased in serum, and TSG-6 and TIMP-2 increased in BALF. IL-1β 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-1β 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.A single intratracheal instillation of MSC reduces the pulmonary and systemic proinflammatory pattern induced by chronic exposure to cigarette smoke in guinea pigs.Not applicable.

Antecedentes: La fístula arteriovenosa autóloga (FAV) es la mejor modalidad de acceso vascular para hemodiálisis por su permeabilidad primaria a largo plazo. Objetivo: Evaluar el mapeo ultrasonográfico preoperatorio y determinar si realizar este ayuda a disminuir el tiempo de maduración, comparado con aquellos a los que no se les realiza. Material y métodos: Se efectúo un estudio retrospectivo, descriptivo y analítico de los pacientes con fístula sin que se hiciera mapeo ultrasonográfico previo con aquellos pacientes que se sometieron a FAV realizando ultrasonido. Resultados: En total se analizaron 104 pacientes, de los cuales 51 correspondió al grupo de USG preoperatorio y 53 en el grupo clínico. No se observaron diferencias significativas entre ambos grupos. El grupo al que se le efectuó ultrasonido, maduraron 35 (33.7%) y en el grupo con solo exploración física maduraron 35 (33.7%). Sin embargo, se demostró que los pacientes a los que se les realizó ultrasonido previo maduraron antes que a los que no. Conclusiones: El ultrasonido preoperatorio demostró ser una herramienta indispensable, ya que permite realizar una mejor planeación y avance en los resultados de las fístulas.

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 (P2X(1), P2X(7), P2Y(4), and P2Y(6)). 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 P2X(7) and P2Y(4), 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 P2X(7) and P2Y(4) receptors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11302-019-09644-7) contains supplementary material, which is available to authorized users.

Background Confinement inside a restricted space causing movement restriction is a stressful condition, potentially leading to spontaneous changes of respiratory parameters that are expected to return to normal values as the stress subsides. Barometric plethysmography is a non-invasive method to study surrogates of pulmonary mechanics in conscious animals enclosed into a plexiglass chamber. This chamber greatly restricts the animal movements but does not cause its immobilization. Methods Respiratory parameters from six rabbits were recorded during 90 min/day for 5 days while the animal was confined inside a plethysmographic chamber. Modifications of respiratory parameters were evaluated by dividing the total length of the recording in three 30-min periods. Results During the 90-min recording, enhanced pause (Penh, a lung resistance surrogate) showed a decreasing trend, coinciding with a decline of the mid-expiratory flow (an airway obstruction surrogate), and time of braking (an end-inspiratory glottis closure surrogate). Respiratory frequency increased from 346 to 363 breaths/min, coinciding with a progressive decline of tidal volume and minute ventilation. Because rabbit responses to stressful situations are predominantly parasympathetic in nature, an increased parasympathetic tone during the first minutes of confinement might explain the initially augmented lung, airways and glottis resistances, and these, in turn, could be responsible for the low initial respiratory frequency. Subsequent changes of these variables probably reflect progressively lower level of stress due to adaptation to the new environment. This pattern did not change in the 5 days studied. Conclusions We concluded that respiratory parameters in rabbits display subtle changes during the first 90 min of movement restriction, probably driven by an initially augmented parasympathetic tone due to stress, with subsequent normalization as stress diminished due to adaptation to the new environment.

The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with β1 integrins (α1β1 and α2β1) in guinea pig. The putative role of β1 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 β1 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 β1 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 β1 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 β1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, β1 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.

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.

Airway smooth muscle (ASM) membrane depolarization through KCl opens L-type voltage dependent Ca2+ channels (Ca(v)1.2); its opening was considered the cause of KCl contraction. This substance is used to bypass intracellular second messenger pathways. It is now clear that KCl also activates RhoA/Rho kinase (ROCK) pathway. ROCK isoforms are characterized as ROCK1 and ROCK2. Because ROCK1 seems the most abundant isotype in lung, we studied its participation in KCl stimulated bovine ASM. With methyl-beta-cyclodextrin (MbetaCD) we disrupted caveolae, a membrane compartment considered as the RhoA/ROCK assembly site, and found that KCl contraction was reduced to the same extent (~26%) as Y-27632 (ROCK inhibitor) treated tissues. We confirmed that KCl induces ROCK activation and this effect was annulled by Y-27632 or MbetaCD. In isolated plasmalemma, ROCK1 was localized in non-caveolar membrane fractions in Western blots from control tissues, but it transferred to caveolae in samples from tissues stimulated with KCl. Ca(v)1.2 was found at the non-caveolar membrane fractions in control and MbetaCD treated tissues. In MbetaCD treated tissues stimulated with KCl, contraction was abolished by nifedipine; only the response to Ca(v)1.2 opening remained as the ROCK component disappeared. Our results show that, in ASM, the KCl contraction involves the translocation of ROCK1 from non-caveolar to caveolar regions and that the proper physiological response depends on this translocation.

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,β-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 P2Y2 and P2Y4 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 P2Y4 and P2Y2 receptors; (2) induces I K increment through activation of the delayed rectifier K+ channels and the high-conductance Ca2+-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.

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.

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 μM 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 μM 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 μM) 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.

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.under physiological conditions, Na(+) permeates through L-VDCC in bovine ASM, probably contributing to sustain membrane depolarization during agonist stimulation.

The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with β1 integrins (α1β1 and α2β1) in guinea pig. The putative role of β1 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 β1 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 β1 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 β1 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 β1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, β1 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.

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 500 μM 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, 500 μM ATP induced inward currents suggestive of P2X1 activation, but higher concentrations (1–5 mM) 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 500 μM 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.

Caffeine has been widely used as a pharmacological tool to evaluate Ca2+ 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 Ca2+ concentration (using Fura–2/AM) were carried out in bovine airway smooth muscle strips during caffeine stimulation. A first stimulation (S1, n=11) with caffeine (10 mM) induced a biphasic change in cytosolic Ca2+, which consisted of a transient Ca2+ 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 ω-conotoxin GVIA (1 μM) and tetrodotoxin (3.1 μM) 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

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.

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.

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H1) and 5-hydroxytryptamine (serotonin) receptors (5-HT2A) 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-β-cyclodextrin (MβCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (∼80%), 5-HT (100%), α-methyl-5-HT (100%), and KCl (∼30%). Cholesterol-loaded MβCD (CL-MβCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (∼85%) and 5-HT (∼59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MβCD and partially reestablished by CL-MβCD. The increase in intracellular Ca2+that was induced by both agonists was reduced by MβCD. The presence of caveolin-1 (Cav-1), H1, 5-HT2A, and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H1receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT2Areceptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H1and 5-HT2Areceptors through their Rho/ROCK signaling.

Airway hyperresponsiveness is a key feature of asthma, but its mechanisms remain poorly understood. Leukotriene D4(LTD4) is one of the few molecules capable of producing airway hyperresponsiveness. In this study, LTD4, but not leukotriene C4(LTC4), produced a leftward displacement of the concentration-response curve to histamine in bovine airway smooth muscle strips. Neither LTC4nor LTD4modified the concentration-response curve to carbachol. In simultaneous measurements of intracellular Ca2+([Ca2+]i) and contraction, histamine or carbachol produced a transient Ca2+peak followed by a plateau, along with a contraction. LTD4increased the histamine-induced transient Ca2+peak and contraction but did not modify responses to carbachol. Enhanced responses to histamine induced by LTD4were 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 LTD4significantly enhanced the phosphorylation induced by histamine, but not by carbachol. L-type Ca2+channel participation in the hyperresponsiveness to histamine was discarded because LTD4did not modify the [Ca2+]ichanges induced by KCl. In tracheal myocytes, LTD4enhanced the transient Ca2+peak induced by histamine (but not by carbachol) and the sarcoplasmic reticulum (SR) Ca2+refilling. Genistein abolished this last LTD4effect. Partial blockade of the SR-ATPase Ca2+pump with cyclopiazonic acid reduced the Ca2+transient peak induced by histamine but not by carbachol. These results suggested that LTD4induces hyperresponsiveness to histamine through activation of the tyrosine kinase pathway and an increasing SR-ATPase Ca2+pump activity. L-type Ca2+channels seemed not to be involved in this phenomenon.

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, P0.05), implicating the neurotransmitters release involvement in the response to caffeine. A similar effect (P0.01) 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), P0.02) 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.

Background In airway smooth muscle (ASM), Ca 2 + influx in response to the Ca 2 + depletion of the sarcoplasmic reticulum (SR) seems to play a role in the regulation of intracellular free Ca 2 + concentrations ([Ca 2 +] i ). This study evaluates some possible Ca 2 + entry pathways activated during SR-Ca 2 + depletion induced by 10 mM caffeine. Methods Enzymatically dispersed bovine ASM cells were loaded with Fura-2/AM to permit measurement of [Ca 2 +] i changes in single cells. Results Caffeine (10 mM) induced a transient increase in [Ca 2 +] i that depleted SR-Ca 2 + content. After caffeine washout, a decrease in basal [Ca 2 +] i (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 μM). External Ca 2 + removal in depolarized and nondepolarized cells induced a decrease in basal [Ca 2 +] i that continued until depletion of the SR-Ca 2 + content. The decrease in [Ca 2 +] i induced by Ca 2 +-free physiological saline solution (PSS) was accelerated in caffeine-stimulated cells. Recovery from undershoot was not observed in Ca 2 +-free PSS. Depolarization with KCl and addition of D600 (30 μM) did not modify recovery. Similar results were obtained when the Na + /Ca 2 + exchanger was blocked by substituting NaCl with KCl in normal PSS (Na + -free PSS) or by adding benzamil amiloride (25 μM). Conclusions SR-Ca 2 + content plays an important role in the Ca 2 + leak induced by Ca 2 +-free medium, and does not depend on membrane potential. Additionally, recovery from undershoot after caffeine depends on extracellular Ca 2 +, and neither voltage-dependent Ca 2 + channels nor the Na + /Ca 2 + exchanger are involved.

Organophosphates are still widely used worldwide and cause thousands of intoxications every year. In this work we investigated the mechanisms of parathion (Pth) airway toxicity, using biochemical and functional approaches. A plethysmographic technique for unrestrained guinea pigs was used to analyze Pth-induced modifications of airway mechanics and responsiveness to acetylcholine (ACh; 0.1–3.2 mg/ml, 2-min inhalation each dose). The isolated perfused rabbit lung preparation was used to study the acute effects of Pth on airway responsiveness to ACh (10–8–10–3 M), histamine (10–8–10–3 M) and substance P (10–10–10–6 M), pulmonary acetylcholinesterase inhibition and cytochrome P450 (P450) activity, and their modifications with previous administration of Pth (1 mg/kg s.c. daily, 7 days). We found that: (1) In guinea pigs Pth (3.2–17 mg/kg i.p.) produced a dose-dependent increase in a lung resistance index (iR l), which was greatly reverted (~50%) by salbutamol (2 mg/ml, 2-min inhalation, or 10 µg/kg i.p.). This salbutamol effect was transient (5–10 min), suggesting that this bronchodilator triggered additional obstructive mechanisms. (2) Pth increased the water content in lung parenchyma samples, but not in trachea or bronchi, and augmented the respiratory secretions measured through monosaccharide content in bronchoalveolar lavage. (3) The increase in iR l was greater in female animals, probably due to a higher P450 basal activity, and completely blocked by pharmacological inhibition of P450 with piperonyl butoxide (500 mg/kg i.p.). (4) In male guinea pigs a subclinical dose of Pth (10 mg/kg i.p.) induced airway hyperresponsiveness to ACh. In isolated perfused rabbit lung Pth (10–6 M) produced airway hyperresponsiveness to ACh and histamine, the latter prevented by atropine (10–5 M). (5) Repetitive exposure to subclinical doses (1 mg/kg s.c.) of Pth during 1 week caused ~80% inhibition of P450 activity in rabbits, which was not enough, however, to prevent the functional manifestation of Pth toxicity in the airways.

Prejunctional and postjunctional effects of several ozone (O3) concentrations, including those found in highly polluted cities, were evaluated in guinea pig airways. Animals bred in O3-free conditions were exposed to air or O3 (0.3, 0.6 or 1.2 ppm) during 4 h, and studied 16-18 h later. Tracheal and bronchial rings were studied in organ baths. Electrical field stimulation (EFS) (100 V, 2 ms, 10 s) was given at increasing frequencies (0.25-16 Hz). Some tissues received atropine (2 microM) and/or propranolol (10 microM). Concentration-response curves to carbachol, isoproterenol, nitroprusside, and substance P were constructed. In tracheas, almost all O3 concentrations decreased the relaxation at low EFS frequencies, but had no effect on the propranolol-resistant (i-NANC) relaxation, suggesting that only adrenergic relaxation was affected. This was a prejunctional effect, since O3 did not modify the responses to isoproterenol. Relaxation induced by a nitric oxide (NO) donor, nitroprusside, was not affected by O3, which agrees with the lack of O3-effect on i-NANC system. O3 did not modify the EFS-induced e-NANC contraction in atropine-treated bronchi, nor the contraction caused by exogenous substance P. By contrast, in bronchi without atropine, 1.2 ppm O3 increased the e-NANC contraction induced by the highest EFS (16 Hz). O3 increased the maximum responses to carbachol in tracheas (1.2 ppm) and bronchi (0.6 and 1.2 ppm). In conclusion, we found that: a) O3 decreased adrenergic relaxation in guinea pig tracheas at low EFS frequencies through a prejunctional alteration; b) O3 did not modify the i-NANC relaxation in tracheas, at least the NO-mediated; c) O3 added a cholinergic component to the bronchial slow-phase (e-NANC) contraction evoked by EFS; and d) O3 enhanced the cholinergic responses in trachea and bronchi by a postjunctional mechanism.

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca2+-free medium and the possibility that each agonist uses a different Ca2+ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca2+) and in Ca2+-free medium. Cch and histamine induced an SBC in bronchial tissues in Ca2+-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca2+-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 microM cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SBC. The SBC induced by Cch or histamine in Ca2+-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca2+ from two different sources during the SBC in Ca2+-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca2+ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only.

Ozone (O3)-induced airway hyperresponsiveness in laboratory animals is usually demonstrated through dose-response curves with inhaled or intravenous bronchoconstrictor agonists. However, comparability of these two routes has not been well documented. Thus guinea pig airway responsiveness to ACh and histamine was evaluated 16–18 h after O3 (3 parts/million, 1 h) or air exposure by two plethysmographic methods (spontaneously breathing and mechanically ventilated) and by two administration routes (inhalatory or intravenous). We found that O3 caused airway hyperresponsiveness to intravenous, but not to inhaled, agonists, independent of the plethysmographic method used. Suitability of the inhalatory route to detect airway hyperresponsiveness was corroborated with inhaled ACh after an antigen challenge or extending O3exposure to 3 h. Acetylcholinesterase activity was not modified after O3 exposure in lung homogenates and blood samples. Thus inhaled agonists were less effective to reveal the airway hyperresponsiveness after an acute O3 exposure than intravenous ones, at least for the 1-h exposure to 3 parts/million, and this difference seems not to be related to an O3-induced inhibition of the acetylcholinesterase activity.