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.

Analgesic and antiinflammatory activities of the ethyl acetate fraction of Bidens pilosa (Asteraceae).

Bidens pilosa is an Asteraceae widely used in traditional medicine for the treatment of various ailments including pain and inflammation. The present work was undertaken to assess the analgesic and antiinflammatory properties of the ethyl acetate fraction of methylene chloride/methanol (1:1) extract of leaves of Bidens pilosa at the gradual doses of 50, 100 and 200 mg/kg in mice and rats, respectively. The analgesic properties of Bidens pilosa were investigated using the acetic acid writhing, hot plate, capsaicin and formalin-induced pain models. This was followed by a study of the antiinflammatory properties using carrageenan, dextran, histamine and serotonin to induce acute inflammation in rat hind paw. The extract provided a significant (p < 0.01) reduction in pain induced by all four models of nociception. It also presented significant (p < 0.05) antiinflammatory activity in all four models of acute inflammation. These results show that the ethyl acetate fraction of methylene chloride/methanol (1:1) of Bidens pilosa has both analgesic and antiinflammatory properties. The qualitative analysis of the fraction by the high-performance liquid chromatography (HPLC) fingerprint revealed the presence of two flavonoids, namely quercetin and iso-okanin, known to have antiinflammatory and antinociceptive properties, which could be responsible for the analgesic and antiinflammatory effects observed.

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.

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.

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.

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.

Distribution and role of aspartate in the nervous system of the chaetognath Sagitta.

Cholinergic control of locomotory muscles in chaetognaths is monitored by diffuse transmitter release through layers of collagen fibers that form the connective stratum of the hydroskeleton. Despite the lack of morphologically defined synaptic junctions, the control of locomotor activity in chaetognaths is highly specific and allows complex behavioral patterns. This complexity suggests the existence of neuromediators acting to modulate the effects of the main motor neurotransmitter, acetylcholine, on muscular contraction. Immunocytochemical investigations performed in Sagitta friderici by using antibodies directed against L-aspartate revealed the presence of the amino acid within abundant fiber networks regularly distributed in the head, trunk, and tail and within discrete groups of cell bodies. In addition to known components of the sensory and motor nervous systems, L-aspartate immunoreactivity revealed previously undescribed intraepidermal networks of axonal profiles. With the exception of two giant anterior fibers radiating from the ventral ganglion, L-aspartate-immunoreactive processes were usually thin and varicose, occasionally making an anastomosis. As indicated by electron microscopy, L-aspartate-immunoreactive varicosities apposed to the connective stratum were filled with synaptic-like vesicles but displayed no synaptic differentiation. Physiologic investigations suggested a potent inhibitory effect of L-aspartate on acetylcholine-induced muscle contraction. The wide distribution pattern of immunoreactive profiles suggests an important role of L-aspartate in motor and sensory functions in chaetognaths. Although classified among excitatory amino acids in vertebrates, aspartate may function as an inhibitory modulator of acetylcholine-induced muscle contraction in these enterocoelous gastroneuralians.

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.

Caffeine acting on pregnant rat myometrium: analysis of its relaxant action and its failure to release Ca2+ from intracellular stores.

1. The effect of caffeine on mechanical activity was studied in pregnant rat myometrium. 2. In muscle cells with intact plasmalemmae, caffeine (0.1-50 mM) produced no contraction whatever the experimental conditions. 3. Caffeine (0.1-10 mM) inhibited, in a concentration-dependent manner, contractions induced by electrical stimulation, potassium-rich (60 mM K+) solution, sodium-free solution or oxytocin (22.5 nM). 4. In Ca2(+)-free solution, various substances (oxytocin, sodium orthovanadate and prostaglandin E2) evoked sustained contractions that were suppressed by caffeine (5-10 mM). When caffeine (greater than 5 mM) was applied during Ca2(+)-loading of the tissue (2.1 mM Ca2+, 5 min) in the presence of a K(+)-rich solution, the subsequent transient contraction induced by a short application (10s) of oxytocin (22.5 nM) in Ca-free solution was reduced (63 +/- 3.5% reduction for 20 mM caffeine, n = 4). 5. In saponin-skinned strips, application of caffeine (5-10 mM) during loading of the Ca2(+)-store increased the subsequent contraction induced by myo-inositol 1,4,5 trisphosphate (IP3, 10 microM). Caffeine (10-30 mM) decreased calcium-activated contractions in skinned fibres lacking a functional internal Ca-store. This effect was reduced by the cyclic AMP-dependent protein kinase inhibitor Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp (8 microM). 6. In conclusion, it is suggested that the inability of caffeine to cause spasm of rat myometrium is due to the absence of a caffeine-sensitive calcium-release channel in the sarcoplasmic reticulum. Relaxant effects of caffeine can be explained by mechanisms leading to a decrease in both the cytoplasmic free Ca2+ concentration and the Ca2 +-sensitivity of the contractile machinery.

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.

Maintained contractions of rat uterine smooth muscle incubated in a Ca2+-free solution.

The effects of acetylcholine (10(-4) M), prostaglandin E2 (10(-6) M), vanadate (5 X 10(-4) M) and fluoride (10(-2) M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. All four substances produced maintained contractions which could be initiated repeatedly after exposure to Ca2+-free solution for more than 1 h, without a significant decrease. The largest contractions were obtained with vanadate and the smallest ones with acetylcholine. The tension was usually 7-30% of the control contraction triggered by an action potential in Ca2+ containing solution. Maintained contractions induced by fluoride were unaffected by isoprenaline while those induced by acetylcholine, prostaglandin E2 and vanadate were completely relaxed. Prostaglandin E2- and vanadate-induced contractions were slightly reduced by Na+ removal or by adding Ca2+ antagonists. In contrast, contractions induced by acetylcholine were suppressed in Na+-free solution and largely inhibited in the presence of Ca2+ antagonists. The depolarization induced by acetylcholine in Ca2+-free solution was strongly dependent on the external Na+ concentration. The relationship between the size of the acetylcholine-induced depolarization and the membrane potential (shifted by constant currents) was linear, giving an apparent reversal potential for acetylcholine close to zero potential. In Ca-free solutions and in the presence of atropine, Na+ action potentials of long duration can be evoked which produced contractions of the same order of magnitude as those initiated by acetylcholine-induced depolarizations. 7 These results are consistent with the hypothesis that the maintained contractions in Ca2+-free solutions induced by several stimulants could be related to Ca2+-independent mechanisms (fluoride) or Ca2+ release from an intracellular store. This latter mechanism would include both pharmacomechanical (prostaglandin E2, vanadate) and electromechanical (acetylcholine) coupling.

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.

[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.

Contractions of rat uterine smooth muscle induced by acetylcholine and angiotensin II in Ca2+-free medium.

The effects of acetylcholine (ACh, 10(-4)M) and angiotensin II (Ang II, 10(-6) M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. Both ACh and Ang II produced transient contractions, the amplitude of which can be taken as a measurement of the amount of Ca2+ present in a drug-sensitive Ca2+ store. The degree of filling of this store depended on the external Ca2+ concentration, and on the presence of contractile responses during the Ca2+ loading period. The existence of two pathways (either direct or transcytoplasmic) is suggested for Ca2+ uptake into the internal Ca2+ store. The rate of filling of the Ca2+ store in 2.1 mM-Ca2+-containing solution was faster (time to half-maximal response, t 1/2 = 29 +/- 2.2 s, n = 4) than the rate of depletion in Ca2+-free solution (t 1/2 = 3 +/- 0.3 min, n = 3). The gradual depletion of this store was much slower at 18 degrees C than at 35 degrees C, and in the presence of vanadate which is known to inhibit Ca2+-ATPases. Methoxyverapamil (D600, 10(-6)-10(-5) M) had no appreciable effect on the direct Ca2+ uptake or on the release of Ca2+ from the store by ACh and Ang II. Mn2+ (10(-3) M) completely inhibited the direct pathway to the internal Ca2+ store and also reduced the release of Ca2+. ACh and Ang II induced repetitive depolarizations close to zero potential which did not parallel the transient contractions as a function of the time of perfusion in Ca2+-free solution. Applications of 2 mM EGTA, 135 mM K+ or Ca2+ antagonists which suppressed or reduced the drug-induced depolarizations did not affect appreciably the drug-induced contractions. These results suggest that myometrial cells have an intracellular Ca2+ store sensitive to different stimulus substances. This store is not affected by depolarization of the plasma membrane and is certainly different from that described in voltage-clamp experiments.

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein.

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle. 2. At 25 degrees C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-CIATP (2.7+/-0.5 microM, n=7) >ATP (12.9+/-1.1 microM, n=9) > or =2-MeSATP (21.9+/-4.8 M, n=4). In the presence of alphabeta-methylene ATP (alphabeta-MeATP, 200 microM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited. 3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP. 4. The A2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1-100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited. 5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to alphabeta-MeATP (200 microM) but were inhibited by ZM 241385 (100 nM). 6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [gamma-32P]-ATP showed that muscular strips rapidly (10-60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57+/-11% (n=3) by 200 microM alphabeta-MeATP. 7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A2A-receptors.

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.

The relaxant action of BRL 34915 in rat uterus.

1 BRL 34915 (0.04-1.3 microM) caused concentration-dependent inhibition of spontaneous phasic spasms of the isolated uterus of the term pregnant rat and this effect was not antagonized by propranolol. Spasms evoked by low concentrations of KCl (less than 20 mM) were inhibited by BRL 34915 but those evoked by higher concentrations (greater than 40 mM) were unaffected. 2 In experiments using extracellular electrical recording, BRL 34915 (10 microM) selectively inhibited oxytocin-induced phasic spasms and the associated spike activity but had little effect on the tonic component of the spasms. BRL 34915, as an inhibitor of phasic spasms to oxytocin (0.2 nM), was antagonized by procaine (0.3 and 1 mM). 3 BRL 34915 (10 microM) did not inhibit Ca2+-induced spasm of saponin-skinned thin myometrial strips. 4 Intracellular microelectrode recording from myometrial strips showed that BRL 34915 (10 microM) inhibited action potentials and phasic spasms in the presence of oxytocin (0.2 nM) and produced a hyperpolarization of 5 mV. 5 In single myometrial cells under current or voltage clamp, BRL 34915 (10 microM) had no effect on action potentials and inward current in Ca2+- or Ba2+-containing media in the presence of tetraethylammonium, 4-aminopyridine and caesium chloride. In the absence of these K+-channel inhibitors, BRL 34915 had no effect on resting membrane potential, membrane resistance, action potential, inward current or outward current. 6 BRL 34915 (1 or 10 microM) had no effect on 86Rb efflux from myometrial strips. 86Rb efflux was increased by oxytocin (0.2 and 20 nM). 7 The relaxant profile of BRL 34915 in the rat uterus is similar to that described for other smooth muscles where an action to open membrane K+-channels has been proposed. BRL 34915 inhibited spike production but produced only a small hyperpolarization without a detectable increase in 86Rb efflux. Membrane resistance and transmembrane currents were unaffected. These results suggest that in the uterus the effects of BRL 34915 may be restricted to K+-channels involved in the production of pacemaker activity.

Characterisation of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery.

Cyclic nucleotides are involved in the control of pulmonary vascular tone. In the present study, we measured the cyclic nucleotide specific phosphodiesterase (PDE) activity in the media of bovine isolated main pulmonary artery (MPA). Total cAMP- and cGMP-PDE activities were measured in microsomal and cytosolic fractions. Both cyclic nucleotides were hydrolysed in these subcellular fractions at consistently higher rate in the cytosolic than in the microsomal fraction. Using different classes of PDE modulator, at least four PDE isoforms (PDE1, 3, 4 and 5) were identified in these fractions. PDE3 (cilostamide-sensitive), PDE4 (rolipram-sensitive) and PDE5 (zaprinast- and DMPPO-sensitive) isoforms appeared as the main isozymes implicated in the cAMP and cGMP hydrolytic activities. Calcium-camodulin stimulated PDE activity (PDE1) was mainly present in the cytosolic fraction. PDE2, although present, had a lower hydrolytic activity since addition of its specific inhibitor, erythro-9-(2-hydroxy-3nonyl)adenine (EHNA), to a combination of inhibitors of PDE3, 4 and 5 produced no further significant reduction in the enzymatic activity. Resolution of PDE activities from the cytosolic fraction using anion exchange chromatography confirmed this finding. Functional experiments performed in endothelium-denuded rings of rat MPA revealed that all specific PDE inhibitors used relaxed precontracted vascular smooth muscle preparations in a concentration-dependent manner. The rank order of potency was cilostamide >zaprinast>rolipram>>EHNA. The present study demonstrates the presence in the smooth muscle cells-containing layer of MPA of PDE1, 3, 4 and 5 isoforms and suggests that PDE3, 4 and 5 are the main enzymes involved in the control of vascular tone.

Selected contribution: effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells.

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca(2+) signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at -60 mV, ACh (0.1-10 microM) induced a concentration-dependent inward current, which, in approximately 50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 microM). Exposure to acrolein (0.2 microM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 microM) concentration-induced initial peak of current (318.8 +/- 28.3 vs. 251.2 +/- 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 microM), the frequency at which subsequent peaks occurred was significantly increased (13.2 +/- 1.1 vs. 8.7 +/- 2 min(-1); n = 20, P < 0.05). ACh-induced current was identified as a Ca(2+)-activated Cl(-) current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca(2+) release, did not alter caffeine-induced transient membrane currents (595 +/- 45 and 640 +/- 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca(2+) concentration response and that the protective role of inhibitors of Cl(-) channels in air pollutant-induced airway hyperresponsiveness should be examined.

Selected contribution: tryptase-induced PAR-2-mediated Ca(2+) signaling in human airway smooth muscle cells.

Tryptase, the major mast cell product, is considered to play an important role in airway inflammation and hyperresponsiveness. Tryptase produces different, sometimes opposite, effects on airway responsiveness (bronchoprotection and/or airway contraction). This study was designed to examine the effect of human lung tryptase and activation of protease-activated receptor (PAR)-2 by synthetic activated peptide (AP) SLIGKV-NH(2) on Ca(2+) signaling in human airway smooth muscle (HASM) cells. Immunocytochemistry revealed that PAR-2 was expressed by HASM cells. Tryptase (7.5--30 mU/ml) induced a concentration-dependent transient relative rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) that reached 207 +/- 32 nM (n = 10) measured by indo 1 spectrofluorometry. The protease inhibitors leupeptin or benzamidine (100 microM) abolished tryptase-induced [Ca(2+)](i) increase. Activation of PAR-2 by AP (1-100 microM) also induced a concentration-dependent transient rise in [Ca(2+)](i), whereas the reverse peptide produced no effect. There was a homologous desensitization of the [Ca(2+)](i) response on repeated stimulation with tryptase or AP. U-73122, a specific phospholipase C (PLC) antagonist, xestospongin, an inositol trisphosphate (IP(3))-receptor antagonist, or thapsigargin, a sarcoplamic Ca(2+)-ATPase inhibitor, abolished tryptase-induced [Ca(2+)](i) response, whereas Ca(2+) removal, in the additional presence of EGTA, had no effect. Calphostin C, a protein kinase C inhibitor, increased PAR-2 [Ca(2+)](i) response. Our results indicate that tryptase activates a [Ca(2+)](i) response, which appears as PAR-2 mediated in HASM cells. Signal transduction implicates the intracellular Ca(2+) store via PLC activation and thus via the IP(3) pathway. This study provides evidence that tryptase, which is increasingly recognized as an important mediator in airway inflammation and hyperresponsiveness, is also a potent direct agonist at the site of airway smooth muscle.