In vitro study of carbon black nanoparticles on human pulmonary artery endothelial cells: effects on calcium signaling and mitochondrial alterations.

Human exposure to manufactured nanoparticles (NPs) is a public health concern. Endothelial cells lining the inner surface of arteries could be one of the primary targets for inhaled nanoparticles. Moreover, it is well known that alteration in calcium signaling is a critical event involved in the physiopathology of cardiovascular diseases. The objective of this study was to assess the role of oxidative stress in carbon black FW2 NPs-induced alteration in calcium signaling and mitochondria in human pulmonary artery endothelial cells. To this end, cells were exposed for 4 or 24 h to FW2 NPs (1-10 µg/cm2) and the following endpoints were studied: (i) production of ROS by fluorimetry and electron paramagnetic resonance, (ii) variation in intracellular calcium concentration by confocal microscopy, and (iii) mitochondrial alteration and apoptosis by confocal microscopy and transmission electronic microscopy. Exposure to FW2 NPs concentration-dependently increases oxidative stress, evidenced by the production of superoxide anion leading to an alteration in calcium content of intracellular organelles, such as endoplasmic reticulum and mitochondria activating, in turn, intrinsic apoptosis. This study provides evidence that FW2 NPs exposure impairs calcium signaling and mitochondria triggered by oxidative stress, and, thus, could act as a cardiovascular disease risk owing to the key role of calcium homeostasis in the control of vascular tone.

Variability of average SUV from several hottest voxels is lower than that of SUVmax and SUVpeak.

OBJECTIVES: To assess variability of the average standard uptake value (SUV) computed by varying the number of hottest voxels within an (18)F-fluorodeoxyglucose ((18)F-FDG)-positive lesion. This SUV metric was compared with the maximal SUV (SUV(max): the hottest voxel) and peak SUV (SUV(peak): SUV(max) and its 26 neighbouring voxels). METHODS: Twelve lung cancer patients (20 lesions) were analysed using PET dynamic acquisition involving ten successive 2.5-min frames. In each frame and lesion, average SUV obtained from the N = 5, 10, 15, 20, 25 or 30 hottest voxels (SUV(max-N)), SUV(max) and SUV(peak) were assessed. The relative standard deviations (SDrs) from ten frames were calculated for each SUV metric and lesion, yielding the mean relative SD from 20 lesions for each SUV metric (SDr(N), SDr(max) and SDr(peak)), and hence relative measurement error and repeatability (MEr-R). RESULTS: For each N, SDr(N) was significantly lower than SDr(max) and SDr(peak). SDr(N) correlated strongly with N: 6.471 × N(-0.103) (r = 0.994; P < 0.01). MEr-R of SUV(max-30) was 8.94-12.63% (95% CL), versus 13.86-19.59% and 13.41-18.95% for SUV(max) and SUV(peak) respectively. CONCLUSIONS: Variability of SUV(max-N) is significantly lower than for SUV(max) and SUV(peak). Further prospective studies should be performed to determine the optimal total hottest volume, as voxel volume may depend on the PET system. KEY POINTS: • PET imaging provides functional parameters of (18) F-FDG-positive lesions, such as SUVmax and SUVpeak. • Averaging SUV from several hottest voxels (SUVmax-N) is a further SUV metric. • Variability of SUVmax-N is significantly lower than SUVmax and SUVpeak variability. • SUVmax-N should improve SUV accuracy for predicting outcome or assessing treatment response. • An optimal total hottest volume should be determined through further prospective studies.

Effects of FW2 Nanoparticles Toxicity in a New In Vitro Pulmonary Vascular Cells Model Mimicking Endothelial Dysfunction.

Several epidemiological studies have revealed the involvement of nanoparticles (NPs) in respiratory and cardiovascular mortality. In this work, the focus will be on the effect of manufactured carbon black NPs for risk assessment of consumers and workers, as human exposure is likely to increase. Since the pulmonary circulation could be one of the primary targets of inhaled NPs, patients suffering from pulmonary hypertension (PH) could be a population at risk. To compare the toxic effect of carbon black NPs in the pulmonary circulation under physiologic and pathological conditions, we developed a new in vitro model mimicking the endothelial dysfunction and vascular dynamics observed in vascular pathology such as PH. Human pulmonary artery endothelial cells were cultured under physiological conditions (static and normoxia 21% O2) or under pathological conditions (20% cycle stretch and hypoxia 1% O2). Then, cells were treated for 4 or 6 h with carbon black FW2 NPs from 5 to 10 µg/cm2. Different endpoints were studied: (i) NPs internalization by transmission electronic microscopy; (ii) oxidative stress by CM-H2DCFDA probe and electron paramagnetic resonance; (iii) NO (nitrites and nitrates) production by Griess reaction; (iv) inflammation by ELISA assay; and (v) calcium signaling by confocal microscopy. The present study characterizes the in vitro model mimicking endothelial dysfunction in PH and indicates that, under such pathological conditions, oxidative stress and inflammation are increased along with calcium signaling alterations, as compared to the physiological conditions. Human exposure to carbon black NPs could produce greater deleterious effects in vulnerable patients suffering from cardiovascular diseases.

[Involvement of the Piezo1 and TRPV4 stretch-activated channels in pulmonary hypertension]. / Implication des canaux mécanosensibles Piezo1 et TRPV4 dans l'hypertension pulmonaire.

Pulmonary hypertension is a pulmonary circulation pathology characterized by remodelling and hyperreactivity of the pulmonary arteries. Vasodilatation/vasoconstriction balance is modified in favour of constriction via, among other things, the proliferation of smooth muscle cells and the development of endothelial dysfunction. In addition, the pulmonary arteries undergo modification of mechanical forces, inducing modified activation of stretch-activated channels (SAC) such as Piezo1 and TRPV4. These ionic channels are sensitive to stretch and their activation can induce various cellular physiological responses, which strongly contribute to development and continuation of the pathology.

Pathophysiology of bronchial smooth muscle remodelling in asthma.

Whereas the role of bronchial smooth muscle remains controversial in healthy subjects its role is well established in asthmatics. Bronchial smooth muscle contraction induces airway narrowing. The smooth muscle also contributes to bronchial inflammation by secreting a range of inflammatory mediators, recruiting and activating inflammatory cells, such as mast cells or T-lymphocytes. In addition, bronchial smooth muscle mass is significantly increased in asthma. Such an increase has been related to a deposition of extracellular matrix proteins, and an increase in both cell size and number. However, the mechanisms of this smooth muscle remodelling are complex and not completely understood. The article will review recent data regarding the pathophysiology of bronchial smooth muscle remodelling in asthma.

Mast cell adhesion to bronchial smooth muscle in asthma specifically depends on CD51 and CD44 variant 6.

BACKGROUND: Mast cells infiltrate the bronchial smooth muscle (BSM) in asthmatic patients, but the mechanism of mast cell adhesion is still unknown. The adhesion molecules CD44 (i.e. hyaluronate receptor) and CD51 (i.e. vitronectin receptor) are widely expressed and bind to many extracellular matrix (ECM) proteins. The aims of the study are (i) to identify the role of ECM in mast cell adhesion to BSM and (ii) to examine the role of CD51 and CD44 in this adhesion. METHODS: Human lung mast cells, human mast cell line (HMC-1), and BSM cells from control donors or asthmatic patients were cultured in the presence/absence of various cytokines. Mast cell-BSM interaction was assessed using (3)H-thymidine-pulsed mast cells, confocal immunofluorescence, or electron microscopy. Adhesion molecules expression and collagen production on both cell types were evaluated by quantitative RT-PCR, western blot, and flow cytometry. RESULTS: Mast cell adhesion to BSM cells mostly involved type I collagen of the ECM. Such an adhesion was increased in normal BSM cells under inflammatory condition, whereas it was maximal in asthmatic BSM cells. Blockade of either CD51 or CD44 significantly decreased mast cell adhesion to BSM. At the molecular level, protein and the transcriptional expression of type I collagen, CD51 or CD44 remained unchanged in asthmatic BSM cells or in mast cells/BSM cells under inflammatory conditions, whereas that of CD44 variant isoform 6 (v6) was increased. CONCLUSIONS: Mast cell-BSM cell adhesion involved collagen, CD44, and CD51, particularly under inflammatory conditions. CD44v6 expression is increased in asthmatic BSM cells.

Signalling pathways involved in the contractile response to 5-HT in the human pulmonary artery.

Serotonin (5-hydroxytryptamine; 5-HT) is a potent pulmonary vasoconstrictor and mitogenic agent whose plasma level is increased in pulmonary hypertensive patients. Thus, we explored the signalling pathways involved in the contractile response to 5-HT in human pulmonary arteries (HPAs). Intact and beta-escin permeabilised rings from HPAs mounted in an organ bath system were used to assess both tension and myofilament Ca(2+)-sensitisation. Microspectrofluorimetry was used for intracellular Ca(2+) recordings in cultured HPA smooth muscle cells. Voltage-operated Ca(2+) channel blockers (nitrendipine and nifedipine) partially reduced the contraction to 5-HT. Thapsigargin or cyclopiazonic acid (CPA), known to deplete sarcoplasmic reticulum Ca(2+) stores, also partially inhibited the contraction, whereas removal of extracellular Ca(2+) under these conditions further inhibited the contraction. Changing from Ca(2+)-free to Ca(2+) containing solution, in the presence of nitrendipine and CPA, a protocol known to stimulate store-operated Ca(2+) channels, induced HPA contractions that were blocked by nickel. Nickel or gadolinium also reduced the contraction to 5-HT. Finally, 5-HT increased intracellular Ca(2+) responses in cultured HPA smooth muscle cells and myofilament Ca(2+)-sensitisation in HPA rings. Collectively, these results indicate that voltage-operated and voltage-independent Ca(2+) channels, as well as Ca(2+) release and myofilament Ca(2+)-sensitisation, participate in 5-HT-induced contraction in HPAs.

Correction to: Comparison of three-parameter kinetic model analysis to standard Patlak's analysis in 18F-FDG PET imaging of lung cancer patients.

Following publication of the original article [1], the authors flagged that the author affiliations detailed in the article are incorrect for the authors M. L. Calcagni and A. Giordano.

Heterogeneity in 5-HT-induced contractile and proliferative responses in rat pulmonary arterial bed.

AIMS: 5-Hydroxytryptamine (5-HT) is a potent vasoconstrictor and mitogen in the pulmonary vascular bed which exhibits phenotypical and functional heterogeneity according to size of the vessels. METHODS: We thus investigated both contractile response and smooth muscle cell (SMC) proliferation in response to 5-HT in rat main extrapulmonary artery (MPA) and intrapulmonary arteries of the first and second order (IPA1 and IPA2). RESULTS: The contractile effect of 5-HT was higher in IPA1 and IPA2 compared to MPA. 5-HT2 receptor antagonists like ketanserin and ritanserin and a 5-HT(1B/D) receptor antagonist, GR127935, partially inhibited the contraction. alpha-Methyl-5-HT, a 5-HT2 receptor agonist, induced a higher contraction in MPA than in IPA and inversely 5-carboxamidotryptamine, a 5-HT1 receptor agonist, induced a higher contraction in IPA2 than in MPA and IPA1. Nitrendipine reduced the contraction, whereas the addition of thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca-ATPases, had an additive blocking effect only in IPA1. The residual contraction to 5-HT was abolished by Y-27632, a rho kinase inhibitor. Finally, SMC proliferation in response to 5-HT was higher in MPA than in IPA2. CONCLUSION: Our results demonstrate regional differences in SMC proliferation as well as in the functional role of 5-HT receptors and the sarcoplasmic reticulum in the contraction.

Comparison of three-parameter kinetic model analysis to standard Patlak's analysis in 18F-FDG PET imaging of lung cancer patients.

BACKGROUND: Patlak's graphical analysis can provide tracer net influx constant (Ki) with limitation of assuming irreversible tracer trapping, that is, release rate constant (kb) set to zero. We compared linear Patlak's analysis to non-linear three-compartment three-parameter kinetic model analysis (3P-KMA) providing Ki, kb, and fraction of free 18F-FDG in blood and interstitial volume (Vb). METHODS: Dynamic PET data of 21 lung cancer patients were retrospectively analyzed, yielding for each patient an 18F-FDG input function (IF) and a tissue time-activity curve. The former was fitted with a three-exponentially decreasing function, and the latter was fitted with an analytical formula involving the fitted IF data (11 data points, ranging 7.5-57.5 min post-injection). Bland-Altman analysis was used for Ki comparison between Patlak's analysis and 3P-KMA. Additionally, a three-compartment five-parameter KMA (5P-KMA) was implemented for comparison with Patlak's analysis and 3P-KMA. RESULTS: We found that 3P-KMA Ki was significantly greater than Patlak's Ki over the whole patient series, + 6.0% on average, with limits of agreement of ± 17.1% (95% confidence). Excluding 8 out of 21 patients with kb > 0 deleted this difference. A strong correlation was found between Ki ratio (=3P-KMA/Patlak) and kb (R = 0.801; P < 0.001). No significant difference in Ki was found between 3P-KMA versus 5P-KMA, and between 5P-KMA versus Patlak's analysis, with limits of agreement of ± 23.0 and ± 31.7% (95% confidence), respectively. CONCLUSIONS: Comparison between 3P-KMA and Patlak's analysis significantly showed that the latter underestimates Ki because it arbitrarily set kb to zero: the greater the kb value, the greater the Ki underestimation. This underestimation was not revealed when comparing 5P-KMA and Patlak's analysis. We suggest that further studies are warranted to investigate the 3P-KMA efficiency in various tissues showing greater 18F-FDG trapping reversibility than lung cancer lesions.

Involvement of oxidative stress and calcium signaling in airborne particulate matter - induced damages in human pulmonary artery endothelial cells.

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H2DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O2-) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM2.5 leads to an intrinsic free radical production (ROS, O2-) and a 4h-exposure to PM2.5 (5-15µg/cm2), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2- levels. The basal intracellular calcium ion level [Ca2+]i was also increased after 4h-exposure to PM2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM2.5 is closely correlated to an increase of oxidative stress.

Paradoxal effect of salbutamol in an in vitro model of bronchoprotection.

Salbutamol-induced hyperresponsiveness to acetylcholine was investigated in human and guinea-pig isolated airways and cultured human airway smooth muscle cells. Salbutamol (10(-7)-10(-5) m) inhibited contractions induced by low concentrations of acetylcholine (10(-8)-10(-7) m) but potentiated contractions induced by higher concentrations of acetylcholine (10(-5)-10(-3) m). Pretreatment with the calcium channel antagonist nicardipine suppressed salbutamol-induced hyperresponse. Stimulation of cultured human airway smooth muscle cells with salbutamol (10(-6) m) amplified intracellular calcium concentration rise induced by acetylcholine (10(-5) m). Propranolol (10(-7) m), a beta1- and beta2-adrenoceptor antagonist, and ICI 118551 (10(-7)-10(-6) m), a beta2-adrenoceptor antagonist, suppressed the inhibitory effect of salbutamol but did not inhibit the hyperresponse on high concentrations of acetylcholine. In contrast, higher concentration of propranolol (10(-6) m) inhibited salbutamol-induced hyperreactivity. Effects of salbutamol were not affected by atenolol, a beta1-adrenoceptor blocker. Salbutamol-induced hyperresponsiveness is mediated through a mechanism involving calcium channel activation.

NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle.

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca(2+)](i)and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 microM indo-lacetoxymethyl ester for 30 min, short (40-60 s) application of ATP (100 microM) or ET-1 (0.1 microM) induced 3-6 cyclic rises in [Ca(2+)](i)(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 microM) for 10 min had no effect on the resting [Ca(2+)](i)value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100-500 microM). SNP (0.001-100 microM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 microM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 microM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca(2+)](i)oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 microM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca(2+)](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication.

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.

Diosmin-induced increase in sensitivity to Ca2+ of the smooth muscle contractile apparatus in the rat isolated femoral vein.

The effect of diosmin, a flavone derivative, on the Ca2+ sensitivity of the venous contractile apparatus was investigated in chemically (beta-escin) skinned strips from the rat isolated femoral vein. Diosmin (0.5-10 microM) shifted to the left the concentration-response curve to Ca2+ (0.05-5 microM). The maximal effect was observed in the presence of 1 microM diosmin which increased the contractile response evoked by 0.15 microM Ca2+ from 26.3% to 78.9% of the maximal Ca(2+)-induced response. This work demonstrates that the venotonic action of diosmin involves an increase in the Ca2+ sensitivity of the contractile apparatus. Such a mechanism of action could represent a new and important means of therapeutic control of vasomotor activity.

Contraction of vascular smooth muscle induced by phorbol 12,13 dibutyrate in human and rat pulmonary arteries.

1. The effect of phorbol 12,13 dibutyrate (PDB) on vascular tone was studied in both human and rat isolated pulmonary arterial strips (HPA and RPA, respectively). 2. PDB (1 nM to 2 microM) produced slowly developing, sustained and concentration-dependent contractions in HPA (mean EC50 = 3.5 nM, n = 5) and RPA (mean EC50 = 120 nM, n = 5). The maximal response was 185.6 +/- 25% and 207 +/- 27.5% (n = 5) of that induced by K(+)-rich (80mM) solution, and 223 +/- 34.5% and 176.5 +/- 38.6% of the noradrenaline (10 microM)-induced contraction in HPA and RPA, respectively. 3. PDB-induced contractions were not altered either by the presence of atropine (10 microM), propranolol (5 microM), phentolamine (5 microM) or tetrodotoxin (10 microM) in the bathing solution, or by the removal of endothelium from pulmonary arteries. 4. In HPA, the amplitude of PDB-induced contractions was significantly reduced by removal of external calcium ions, addition of verapamil (10 microM) or trifluoperazine (TFP, 5 microM) and significantly increased by Bay K 8644 (0.5 microM). In contrast, in RPA, calcium-free solution and verapamil had only a moderate effect on the maximal PDB-induced contraction (approximately 20% reduction), whereas Bay K 8644 and TFP had no significant effect. In both HPA and RPA, PDB-contractions in calcium-free solutions were not modified by ryanodine (25 microM) or by 8-(N,N diethylamino)octyl-3,4,5, trimethoxybenzoate hydrochloride (TMB-8, 50 microM). 5. PDB-induced contractions were inhibited by protein kinase C (PKC) antagonists. The maximal response was decreased by 60 +/- 10.5% and 35 +/- 11.5% (n = 5) by 145-isoquinolinesulphonyl)-2-methylpiperazine (H7, 50 microM), 70.5 +/- 12.2% and 56 +/- 18% (n = 5) by phloretin (100 microM) and 80.7 +/- 8.4% and 71 +/- 14% (n = 5) by staurosporine (25 nM) in HPA and RPA, respectively.6. Long term treatment (15-20 h) of arterial strips with phorbol esters (phorbol 12,13 didecanoate, or PDB) abolished the contractile response to subsequent addition of PDB.7. These results show that PDB is a potent vasoconstrictor agent in human and rat pulmonary arteries. Unlike the rat, part of the PDB response depends on calcium influx in human preparations. PDB action appears mainly mediated by the activation of protein kinase C. PKC could thus play a major role in the control of vascular pulmonary reactivity.

Effect of passive sensitization on the mechanical activity of human isolated bronchial smooth muscle induced by substance P, neurokinin A and VIP.

1. The effect of passive sensitization on the mechanical activity of human isolated bronchial smooth muscle induced by the following neuropeptides substance P (SP), neurokinin A (NKA) and vasoactive intestinal peptide (VIP) was studied both in the absence and in the presence of the neutral endopeptidase (NEP) inhibitor, phosphoramidon. 2. Cumulative concentration-response curves (CCRC) to these neuropeptides were constructed in human passively sensitized isolated bronchial rings and compared to those in paired controls. Passively sensitized human isolated bronchial rings were tissues incubated overnight in serum from asthmatic patients atopic to Dermatophagoides pteronyssinus and paired controls were tissues originating from the same lung specimens but incubated overnight in serum from healthy donors. 3. In the absence of phosphoramidon, passive sensitization significantly increased the amplitude of the contractile responses to SP and NKA including that to the maximal concentration given from 50 +/- 5% to 76 +/- 6% (n = 5, P < 0.05) and from 70 +/- 7% to 101 +/- 6% (n = 5, P < 0.05) of the maximal response to acetylcholine, respectively. Passive sensitization significantly shifted to the left the CCRC for both tachykinins as measured by the geometric means dose-ratios which were 8.5 (95% confidence limits (CL): 3.1-13.9) and 7.3 (95% CL: 4.2-10.3) for SP and NKA, respectively. 4. In the presence of phosphoramidon (10 microM), passive sensitization still increased significantly the amplitude of the contractile responses to SP and NKA including that to the maximal concentration given from 74 +/- 4% to 115 +/- 7% (n = 5, P<0.05) and from 104 +/- 9% to 146 +/- 16% (n = 5, P<0.05)of the maximal response to acetylcholine, respectively. Passive sensitization still significantly shifted to the left the CCRC for both tachykinins as measured by the dose-ratios which were 9.0 (95% CL:4.3-13.6) and 5.4 (95% CL: 2.9-7.9) for SP and NKA, respectively.5. The relaxant response to the maximal concentration of VIP given in tissues precontracted with histamine (0.5 mM) was significantly reduced by passive sensitization from 41 +/- 4% to 25 +/- 3% (n = 5,P <0.05) of the amplitude of the precontraction in the absence of phosphoramidon and from 72 +/- 1%to 49 +/- 4% (n = 5, P<0.05) in the presence of phosphoramidon (10 microM). Passive sensitization significantly shifted to the right the CCRC for VIP as measured by the dose-ratios which were 10.4(95% CL: 6.6-14.1) and 6.4 (95% CL: 3.0-9.8) in the absence and in the presence of phosphoramidon,respectively.6. We conclude that passive sensitization enhances the mechanical response to neuropeptides which contract human isolated bronchial smooth muscle and reduces that to a neuropeptide which relaxes it.The mechanism of passive sensitization-induced changes in the mechanical activity appears to be independent of a decrease in NEP activity since these changes persist in the presence of the NEP inhibitor, phosphoramidon.

Role of EDHF in the vasodilatory effect of loop diuretics in guinea-pig mesenteric resistance arteries.

1. Relaxing effect of loop diuretics, piretanide and furosemide in comparison with acetylcholine (ACh) was investigated in guinea-pig isolated mesenteric resistance arteries. 2. Concentration-response curves to ACh (0.001 - 10 microM) and diuretics (0.0001 - 1 microM) were constructed in noradrenaline (10 - 30 microM)-precontracted arteries incubated either in normal physiological salt solution (PSS) or in 30 mM KCl PSS (K-PSS). 3. In PSS, maximal relaxations (R(max)) and pD(2) to ACh were 87+/-2% and 7.1+/-0.1 (n=10). L-N(G)-nitro-arginine methyl ester (L-NAME, 100 microM) reduced R(max) by 20% (P<0.01, n=7) and pD(2) by 10% (P<0.01). In contrast, indomethacin (10 microM) increased R(max) by 19% (P<0.01, n=8) and pD(2) by 10% (P<0.05). Combination of L-NAME+indomethacin reversed the effect observed with either of these inhibitors used alone. In K-PSS, R(max) was attenuated by 40% (P<0.001, n=6) compared to PSS. L-NAME reduced R(max) by 65% (P<0.01, n=5) and increased pD(2) by 15 fold. L-NAME+indomethacin suppressed the resistant relaxation. 4. In PSS+L-NAME+indomethacin, inhibitors of small (SK(Ca); apamin, 0.1 microM) and large (BK(Ca); iberiotoxin and charybdotoxin, 0.1 microM) conductance Ca(2+)-sensitive K(-)-channels used alone had little effect on the ACh-response. Combination of apamin+iberiotoxin reduced R(max) by 40% (P<0.05, n=7) while apamin+charybdotoxin fully abolished the resistant relaxation. 5. In PSS, piretanide and furosemide induced relaxation with R(max): 89+/-3% vs 84+/-5% and pD(2): 8.5+/-0.1 vs 7.7+/-0.2 (P<0.01) for piretanide (n=11) and furosemide (n=10), respectively. Endothelial abrasion suppressed relaxation to diuretics. L-NAME and indomethacin used alone or in combination did not significantly modify the response to diuretics. 6. In K-PSS, piretanide-induced relaxation was abolished whereas that to furosemide was reduced by 70% (P<0.001, n=9) compared to PSS and was suppressed by L-NAME+indomethacin. In PSS+L-NAME+indomethacin, apamin slightly reduced relaxation to diuretics whereas charybdotoxin or iberiotoxin abolished the response. 7. These results indicate that ACh-evoked relaxation is mediated by both NO/PGl(2)-dependent and -independent mechanisms. The EDHF-dependent component relies on activation of Ca(2+)-activated K(+) channels, is sensitive to a combination of apamin+charybdotoxin and to a smaller degree to a combination of apamin+iberiotoxin. Loop diuretic-induced relaxation is endothelium-dependent, appears to be mediated by NO, PGl(2) and EDHF for furosemide and EDHF only for piretanide. For the two diuretics, opening of BK(Ca) channels may be involved in the relaxation.

[Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity.

1. Cytosolic calcium concentration ([Ca2+]i) by indo 1 microspectrofluorimetry in freshly isolated cells and isometric contraction of isolated rings were measured in response to muscarinic cholinoceptor stimulation in rat tracheal smooth muscle. 2. In isolated myocytes, acetylcholine (ACh, 0.03-1 microM) caused a rapid and graded increase in [Ca2+]i up to a net amplitude of 492 +/- 26 nM (n = 19) which gradually declined. The EC50 for ACh was 0.13 microM. This first [Ca2+]i peak was followed, when the ACh concentration increased, in approximately 50-60% of the cells, by successive peaks of decreased amplitude ([Ca2+]i oscillations) superimposed on the plateau phase. Whereas the percentage of cells exhibiting [Ca2+]i oscillations remained consistent, the frequency of these oscillations increased to up to 10 min-1 with an ACh concentration of 100 microM. 3. Removal of extracellular calcium (in the presence of EGTA, 0.4 mM) or addition of the voltage-dependent Ca(2+)-channel blocker verapamil (10 microM) did not alter the first [Ca2+]i peak, the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca(2+)-ATPase, thapsigargin (1 microM), completely abolished the [Ca2+]i response. Thapsigargin (1 microM) also blocked the caffeine (5 mM)-induced transient rise in [Ca2+]i. 4. Atropine (a non-selective muscarinic cholinoceptor antagonist) and 4-diphenyl acetoxy N-methyl piperidine (4-DAMP, a selective M3 antagonist) inhibited the [Ca2+]i response to muscarinic cholinoceptor activation with an IC50 of 13 and 20 nM, respectively. Pirenzepine (a selective M1 antagonist) also totally inhibited the [Ca2+]i response to ACh but with a higher IC50 of 2 microM. Methoctramine (a selective M2 antagonist) up to a concentration of 10 microM caused only a 40% inhibition. The effect of muscarinic antagonists on cumulative concentration-response curves (CCRC) for carbachol was assessed at the following concentrations: atropine and 4-DAMP at 3, 10 and 30 nM; pirenzepine 0.3, 1 and 3 microM, and methoctramine at 1, 3 and 10 microM. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA2 values were 9.2, 8.8, 6.7 and 6.3, respectively. 5. In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca2+]i oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca2+]i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca2+]i and the contractile responses are primarily dependent on activation of the M3 receptor subtype.

Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery.

(1) Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). (2) The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 micro M) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 micro M), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 micro M) also inhibited significantly (22%) the cAMP-PDE activity. (3) Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC(50)=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. (4) Sildenafil (0.1 nM-50 micro M) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 micro M). The potency of sildenafil (IC(50)=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC(50)=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 micro M) and H89 (1 micro M), potent inhibitors of PKG and PKA, respectively. (5) In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca(2+)](i) response. (6) This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.