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.

[Nerve growth factor (NGF) in pulmonary hypertension (PH)]. / Le facteur de croissance des nerfs (NGF) dans l'hypertension pulmonaire (HTP).

Pulmonary hypertension (PH) is the main pathology in lung circulation, characterized by increased pressure in pulmonary arteries and ultimately resulting in right heart failure with potentially fatal outcomes. Given the current lack of available curative treatments, it is of paramount importance to identify novel therapeutic targets. Due to its involvement in pulmonary arterial remodeling, hyperreactivity, and inflammation, our explorations have focused on the nerve growth factor (NGF), offering promising avenues for innovative therapeutic approaches.

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.

[The expression and role of nerve growth factor (NGF) in pulmonary hypertension]. / Expression et rôle du facteur de croissance des nerfs NGF dans l'hypertension pulmonaire.

Pulmonary hypertension is a severe multifactorial disease of the pulmonary circulation characterized by a progressive elevation in mean pulmonary arterial pressure (PAPm), leading to right ventricular failure and the death of the patient. Current therapies slow the progression of the disease but do not offer a cure. Nerve growth factor NGF is a growth factor playing a significant role in the pathophysiology of pulmonary hypertension, particularly in pulmonary arterial hyperreactivity, and the remodelling and inflammation of the pulmonary vasculature. Thus, targeting NGF may offer new therapeutic strategies in the treatment of this disease.

Enzyme-assisted mineralization of calcium phosphate: exploring confinement for the design of highly crystalline nano-objects.

In hard tissues of vertebrates, calcium phosphate (CaP) biomineralization is a fascinating process that combines specific physicochemical and biochemical reactions, resulting in the formation of extracellular matrices with elegant nanoarchitectures. Although several "biomimetic" strategies have been developed for the design of mineralized nanostructured biointerfaces, the control of the crystallization process remains complex. Herein, we report an innovative approach to overcome this challenge by generating, in situ, CaP precursors in a confined medium. For this purpose, we explore a combination of (i) the layer-by-layer assembly, (ii) the template-based method and (iii) the heterogeneous enzymatic catalysis. We show the possibility of embedding active alkaline phosphatase in a nanostructured multilayered film and inducing the nucleation and growth of CaP compounds under different conditions. Importantly, we demonstrate that the modulation of the crystal phase from spheroid-shaped amorphous CaP to crystalline platelet-shaped hydroxyapatite depends on the degree of confinement of active enzymes. This leads to the synthesis of highly anisotropic mineralized nanostructures that are mechanically stable and with controlled dimensions, composition and crystal phase. The present study provides a straightforward, yet powerful, way to design anisotropic nanostructured materials, including a self-supported framework, which may be used in broad biomedical applications.

Calcium phosphate mineralization through homogenous enzymatic catalysis: Investigation of the early stages.

HYPOTHESIS: The crystallization of calcium phosphate (CaP) is a ubiquitous process that occurs in several mineralized tissues and involves a variety of biochemical and chemical reactions. This issue has been hitherto continuously studied from supersaturated solutions (chemical procedure), i.e. by adding calcium and orthophosphate ions in a homogenous phase. Yet, both in vivo and in vitro investigations have clearly shown the implication of enzymes, namely alkaline phosphatase (ALP), to initiate the mineralization process by generating orthophosphate ions. EXPERIMENTS: We report a thorough investigation on the mechanism of enzyme-induced mineralization in homogenous phase (enzymatic procedure). For this purpose, ALP is introduced in Ca2+/Mg2+-containing solution (pH = 7.4; 37 °C), and its activity modulated by the concentration of its substrate. FINDINGS: Results show that after 24 h of mineralization both chemical and enzymatic procedures lead to the formation of well-crystalline hydroxyapatite nano-objects, however with noticeable impact on their shape and dimensions. Remarkably enough, by combining in situ monitoring and ex situ characterizations, we identify several intermediate phases, including amorphous phase, dicalcium phosphate dehydrate phase (DCPD or brushite) and Whitlockite (WH). Besides, mineralized nano-objects with a core-shell structure is observed, and hydroxyapatite platelets are shown to grow on the surface of their shell.

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.

Prevention of a hypoxic Ca(2+)(i) response by SERCA inhibitors in cerebral arterioles.

1. The aim of the study was to investigate the mechanism of a novel effect of hypoxia on intracellular Ca(2+) signalling in rabbit cerebral arteriolar smooth muscle cells, an effect that was resistant to the L-type Ca(2+) channel antagonist methoxyverapamil (D600). 2.[Ca(2+)](i) of smooth muscle cells in intact arteriolar fragments was measured using the Ca(2+)-indicator dye fura-PE3. Hypoxia (PO(2) 10 - 20 mmHg) lowered basal [Ca(2+)](i) but did not inhibit Ca(2+) entry pathways measured by Mn(2+)-quenching of fura-PE3. 3. The effect of hypoxia was completely prevented by thapsigargin or cyclopiazonic acid, selective inhibitors of sarcoplasmic reticulum Ca(2+) ATPase (SERCA). Since these inhibitors do not block Ca(2+) extrusion or uptake via the plasma membrane, the data indicate that the effect of hypoxia depends on a functional sarcoplasmic reticulum. 4. Because actions of nitric oxide (NO) on vascular smooth muscle are also prevented by SERCA inhibitors it was explored whether the effect of hypoxia occurred via modulation of endogenous NO release. Residual NOS-I and NOS-III were detected by immunostaining, and there were NO-dependent effects of NOS inhibitors on Ca(2+)(i)-signalling. Nevertheless, inhibition of endogenous NO production did not prevent the effect of hypoxia on [Ca(2+)](i). 5. The experiments reveal a novel nitric oxide-independent effect of hypoxia that is prevented by SERCA inhibitors.

[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.

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.

Expression and function of cystic fibrosis transmembrane conductance regulator in rat intrapulmonary arteries.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cyclic adenosine monophosphate (cAMP)-dependent chloride channel located mainly at the apical membrane of epithelial cells. In myocytes of pulmonary arteries, numerous chloride channels have been identified and described, but not the CFTR. Thus the presence and function of the CFTR was investigated in rat intrapulmonary arteries. CFTR expression, localisation and function were analysed in cultured smooth muscle cells using Reverse transcriptase (RT)-PCR and immunoprecipitation followed by protein kinase A phosphorylation, immunolocalisation and an iodide efflux assay, respectively. The role of the CFTR in pulmonary vasoreactivity was determined in arterial rings using an organ bath system. RT-PCR and immunoprecipitation analyses, as well as the immunolocalisation study, revealed the expression of CFTR gene transcripts and protein. The iodide efflux assay showed the existence of functional cAMP-, calcium- and volume-dependent chloride channels. Furthermore, the following effects were found: 1) inhibition of forskolin/genistein-activated iodide efflux by glibenclamide, diphenylamine-2-carboxylic acid and CFTR-specific inhibitor (CFTR(inh))-172; 2) activation of iodide efflux by the benzoquinolizinium derivative CFTR activators MPB-07 and MPB-91; and 3) inhibition of MPB-dependent efflux by CFTR(inh)-172. Finally, CFTR activators induced concentration-dependent vasorelaxation in rings preconstricted with phenylephrine, in the presence or absence of endothelium. The present results are the first to reveal functional cyclic adenosine monophosphate-regulated cystic fibrosis transmembrane conductance regulator contributing to endothelium-independent vasorelaxation in rat intrapulmonary arterial myocytes.

Positive and negative coupling of the endothelin ETA receptor to Ca2+-permeable channels in rabbit cerebral cortex arterioles.

1. Arteriolar segments were isolated from pial membrane and studied within 10 h. Current-clamp and voltage-clamp measurements were made by patch-clamp recording from smooth muscle cells within arterioles. [Ca2+]i was measured from the smooth muscle cell layer by digital imaging of emission from fura-PE3 which was loaded into arterioles by pre-incubation with the acetoxymethyl ester derivative. The external diameter of arterioles was measured using a video-dimension analyser. 2. Endothelin-1 (ET1) was a potent constrictor of isolated arterioles and induced a sustained depolarization up to -27 mV and reduced membrane resistance (EC50 140-170 pm). At a constant holding potential of -60 mV ET-1 induced a transient followed by a sustained inward current. ET1 inhibited L-type voltage-dependent Ca2+ current. 3. ET1 induced a transient followed by sustained elevation of [Ca2+]i. The sustained effect was dependent on extracellular Ca2+. It occurred at a constant holding potential of -60 mV and was not inhibited by the Ca2+ antagonists nicardipine (1 microM) or D600 (10 microM). Thapsigargin (1 microM) completely depleted Ca2+ from caffeine- and ET1-sensitive sarcoplasmic reticulum but did not inhibit the ET1-induced sustained elevation of [Ca2+]i. ET1 effects on [Ca2+]i were prevented by the ETA receptor antagonist BQ123 (cyclo-D-Asp-Pro-D-Val-Leu-D-Trp). 4. The data suggest that ETA receptors are negatively coupled to L-type Ca2+ channels and positively coupled to receptor-operated Ca2+-permeable channels. Inhibition of L-type Ca2+ channel activity may suppress autoregulation, and Ca2+ influx through receptor-operated channels may have a major functional role in the potent long-lasting constrictor effect of endothelin-1 in the cerebral microcirculation.

Sodium-potassium-ATPase electrogenicity in cerebral precapillary arterioles.

Electrogenicity of the Na(+)/K(+) pump has the capability to generate a large negative membrane potential independently of ion-channel current. The high background membrane resistance of arterioles may make them susceptible to such an effect. Pump current was detected by patch-clamp recording from smooth muscle cells in fragments of arterioles (diameter 24-58 microm) isolated from pial membrane of rabbit cerebral cortex. The current was 20 pA at -60 mV, and the extrapolated zero current potential was -160 mV. Two methods of estimating the effect of pump electrogenicity on resting potential indicated an average contribution of -35 mV. In 20% of the recordings, block of inward rectifier K(+) channels by 10-100 microM Ba(2+) led to a small depolarization, but hyperpolarization was a more common response. Ba(2+) also inhibited depolarization evoked by 20 mM K(+). In arterioles within intact pial membrane, Ba(2+) failed to evoke constriction but inhibited K(+)-induced constriction. The data suggest that cerebral arterioles are vulnerable to the hyperpolarizing effect of the Na(+)/K(+) pump, excessive effects of which are prevented by depolarizing inward rectifier K(+) current

Hepatitis C virus proteins do not directly trigger fibrogenic events in cultured human liver myofibroblasts.

Although liver fibrosis is the major complication of hepatitis C virus (HCV) infection, the mechanisms of fibrogenesis in this setting are not completely understood. The aim of this study was to test the direct effect of HCV proteins on signalling- and fibrosis-related events in cultured human liver myofibroblasts, the effector cells of liver fibrogenesis. Cultured myofibroblasts were exposed to recombinant HCV core, a structural protein, and nonstructural proteins (NS) 3, NS 4 and NS 5. HCV proteins did not significantly increase DNA synthesis in myofibroblasts. We then examined if these proteins affected early signalling events. None of the HCV proteins affected the phosphorylation of the mitogen activated protein kinases/extracellular regulated kinases 1 and 2, or of the phosphatidylinositol 3-kinase target, Akt. HCV proteins had also no effect on intracellular calcium concentration. In other experiments, fibrogenesis-related parameters were measured. None of the HCV proteins had any effect on the secretion of type I collagen, tissue inhibitor of matrix metalloproteinases type 1, gelatinase or urokinase. Alpha-smooth muscle actin expression was also not modified. In summary, our experiments do not support a direct effect of these HCV proteins on fibrogenic cells.