[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.

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.

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.

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.

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells.

BACKGROUND AND PURPOSE: Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor. EXPERIMENTAL APPROACH: Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 µmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 µmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 µmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.

Dehydroepiandrosterone (DHEA) inhibits voltage-gated T-type calcium channels.

BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) and its sulfated form, DHEAS, are the most abundant steroid hormones in the mammalian blood flow. DHEA may have beneficial effects in various pathophysiological conditions such as cardiovascular diseases or deterioration of the sense of well-being. However to date, the cellular mechanism underlying DHEA action remains elusive and may involve ion channel modulation. In this study, we have characterized the effect of DHEA on T-type voltage-activated calcium channels (T-channels), which are involved in several cardiovascular and neuronal diseases. KEY RESULTS: Using the whole-cell patch-clamp technique, we demonstrate that DHEA inhibits the three recombinant T-channels (Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3) expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells. This effect of DHEA is both concentration (IC(50) between 2 and 7µM) and voltage-dependent and results in a significant shift of the steady-state inactivation curves toward hyperpolarized potentials. Consequently, DHEA reduces window T-current and inhibits membrane potential oscillations induced by Ca(V)3 channels. DHEA inhibition is not dependent on the activation of nuclear androgen or estrogen receptors and implicates a PTX-sensitive Gi protein pathway. Functionally, DHEA and the T-type inhibitor NNC 55-0396 inhibited KCl-induced contraction of pulmonary artery rings and their effect was not cumulative. CONCLUSIONS: Altogether, the present data demonstrate that DHEA inhibits T-channels by a Gi protein dependent pathway. DHEA-induced alteration in T-channel activity could thus account for its therapeutic action and/or physiological effects.

Quercetin potentiates insulin secretion and protects INS-1 pancreatic ß-cells against oxidative damage via the ERK1/2 pathway.

BACKGROUND AND PURPOSE: Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic ß-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting ß-cells. Using the INS-1 ß-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on ß-cell dysfunctions induced by hydrogen peroxide (H(2)O(2)). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison. EXPERIMENTAL APPROACH: Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay. KEY RESULTS Quercetin (20 µmol·L(-1)) potentiated both glucose (8.3 mmol·L(-1))- and glibenclamide (0.01 µmol·L(-1))-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L(-1)), protected ß-cell function and viability against oxidative damage induced by 50 µmol·L(-1) H(2)O(2) and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective. CONCLUSION AND IMPLICATIONS: Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected ß-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing ß-cell dysfunction associated with diabetes deserves further investigation.

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells.

T-type Ca(2+) channel family includes three subunits Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3 and have been shown to control burst firing and intracellular Ca(2+) concentration ([Ca(2+)](i)) in neurons. Here, we investigated whether Ca(V)3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca(V)3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca(V)3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca(2+)](i) oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around -61mV. [Ca(2+)](i) oscillations were critically dependent on Ca(2+) influx through Ca(V)3.1 channels and did not involve Ca(2+) release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca(V)3.1 channels. The trigger of the oscillations was the Ca(V)3.1 window current. This current induced continuous [Ca(2+)](i) increase at -60mV that depolarized the cells and triggered MPOs. Shifting the Ca(V)3.1 window current potential range by increasing the external Ca(2+) concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I(h)) was not required to induce MPOs, but when expressed together with Ca(V)3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca(V)3.1 window current is critical in triggering intrinsic electrical and [Ca(2+)](i) oscillations.

Stimulation of L-type Ca2+ channels by inositol pentakis- and hexakisphosphates in rat vascular smooth muscle cells.

The electrophysiological effects of D-myo-inositol 1,3,4,5,6-pentakisphosphate (InsP5) and D-myo-inositol hexakisphosphate (InsP6), which represent the main cellular inositol polyphosphates, were studied on L-type Ca2+ channels in single myocytes of rat portal vein. Intracellular infusion of InsP5 (up to 50 micro M) or 10 micro M InsP6 had no action on Ba2+ current, whereas 50 micro M InsP6 or 10 micro M InsP5 plus 10 micro M InsP6 (InsP5,6) stimulated the inward current. The stimulatory effect of InsP5,6 was also obtained in external Ca2+-containing solution. The stimulated Ba2+ current retained the properties of L-type Ba2+ current and was oxodipine sensitive. PKC inhibitors Ro 32-0432 (up to 500 nM), GF109203X (5 micro M) or calphostin C (100 nM) abolished the InsP5,6-induced stimulation. Neither the PKA inhibitor H89 (1 micro M) nor the protein phosphatase inhibitors okadaic acid (500 nM) or cypermethrin (1 micro M) prevented or mimicked the InsP5,6-induced stimulation of Ba2+ current. However, InsP5 or InsP6 could mimic some effects of protein phosphatase inhibitor so as to extend after washing-out forskolin the stimulatory effects of the adenylyl cyclase activator on Ba2+ current. These results indicate that InsP5 and InsP6 may act as intracellular messengers in modulating L-type Ca2+ channel activity and so could be implicated in mediator-induced contractions of vascular smooth muscle cells.

Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells.

OBJECTIVE: Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS: Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS: No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS: Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

[Is atypical sodium current related to arterial pathophysiology?]. / Un courant sodique atypique lié à la patho-physiologie artérielle?

Primary cultured human coronary myocytes, derived from patients with end-stage heart failure (NYHA, classes III and IV) caused by an ischemic disease and undergoing heart transplantation, express a voltage-gated tetrodotoxin-sensitive sodium current (INa). This current has atypical electrophysiological and pharmacological properties and regulates intracellular sodium ([Na+]i) and calcium ([Ca2+]i). Our work is aimed at identifying its role and regulation of expression during pathophysiology. We currently investigate whether INa is expressed in vascular smooth muscles cells (VSMCs) isolated from either healthy or diseased (atheromatous) arteries in human and, in parallel, in pig, rabbit and rat. Cells were enzymatically isolated, primary cultured and macroscopic INa were recorded using the whole cell patch clamp technique. We found that INa is expressed in VSMCs grown from human aortic (90%; n = 48) and pulmonary (44%; n = 16) arteries and in the human aortic cell line HAVSMC (94%; n = 27). INa was also detected in pig coronary (60%; n = 25) and rabbit aortic (47%; n = 15) VSMCs, but not in rat aortic myocytes (n = 30). These different INa were activated at similar range of potentials (approximately -45 mV), had similar sensitivity to tetrodotoxin (IC50 around 5 nM) and similar density (2 to 4 pA/pF). Their expression was related to cell dedifferentiation in vitro. However, INa was observed more frequently in human myocytes derived from diseased arteries (ischemic cardiopathy) than in those derived from healthy tissues (dilated cardiopathy). In conclusion, INa may contribute to increase the basal arterial contractility and play a role in pathological situations including hypertension.

Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery.

1. Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. 2. In single myocytes, iberiotoxin (0.1 microM)-, charybdotoxin (0.1 microM)-, apamin (0.5 microM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BK(Ca) and SK(Ca)) as well as voltage-dependent potassium channels (K(V)). Charybdotoxin and iberiotoxin inhibited the same population of BK(Ca) but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0. 1 - 25 mM) induced a concentration-dependent inhibition of K(V) without affecting the iberiotoxin- or the apamin-sensitive currents. 3. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. 4. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.

Regulation of Ca2+ homeostasis by atypical Na+ currents in cultured human coronary myocytes.

Primary cultured human coronary myocytes (HCMs) derived from ischemic human hearts express an atypical voltage-gated tetrodotoxin (TTX)-sensitive sodium current (I(Na)). The whole-cell patch-clamp technique was used to study the properties of I(Na) in HCMs. The variations of intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were monitored in non-voltage-clamped cells loaded with Fura-2 or benzofuran isophthalate, respectively, using microspectrofluorimetry. The activation and steady-state inactivation properties of I(Na) determined a "window" current between -50 and -10 mV suggestive of a steady-state Na+ influx at the cell resting membrane potential. Consistent with this hypothesis, the resting [Na+]i was decreased by TTX (1 micromol/L). In contrast, it was increased by Na+ channel agonists that also promoted a large rise in [Ca2+]i. Veratridine (10 micromol/L), toxin V from Anemonia sulcata (0.1 micromol/L), and N-bromoacetamide (300 micromol/L) increased [Ca2+]i by 7- to 15-fold. This increase was prevented by prior application of TTX or lidocaine (10 micromol/L) and by the use of Na(+)-free or Ca(2+)-free external solutions. The Ca(2+)-channel antagonist nicardipine (5 micromol/L) blocked the effect of veratridine on [Ca2+]i only partially. The residual component disappeared when external Na+ was replaced by Li+ known to block the Na+/Ca2+ exchanger. The resting [Ca2+]i was decreased by TTX in some cells. In conclusion, I(Na) regulates [Ca2+]i in primary cultured HCMs. This regulation, effective at baseline, involves a tonic control of Ca2+ influx via depolarization-gated Ca2+ channels and, to a lesser extent, via a Na+/Ca2+ exchanger working in the reverse mode.

Potassium ions and endothelium-derived hyperpolarizing factor in guinea-pig carotid and porcine coronary arteries.

Experiments were designed to determine in two arteries (the guinea-pig carotid and the porcine coronary arteries) whether or not the endothelium-derived hyperpolarizing factor (EDHF) can be identified as potassium ions, and to determine whether or not the inwardly rectifying potassium current and the Na+/K+ pump are involved in the hyperpolarization mediated by EDHF. The membrane potential of vascular smooth muscle cells was recorded with intracellular microelectrodes in the presence of N(omega)-L-nitro-arginine (L-NA) and indomethacin. In vascular smooth muscle cells of guinea-pig carotid and porcine coronary arteries, acetylcholine and bradykinin induced endothelium-dependent hyperpolarizations (-18+/-1 mV, n = 39 and -19+/-1 mV, n = 7, respectively). The hyperpolarizations were not affected significantly by ouabain (1 microM), barium chloride (up to 100 microM) or the combination of ouabain plus barium. In both arteries, increasing extracellular potassium concentration by 5 or 10 mM induced either depolarization or in a very few cases small hyperpolarizations which never exceeded 2 mV. In isolated smooth muscle cells of the guinea-pig carotid artery, patch-clamp experiments shows that only 20% of the vascular smooth muscle cells expressed inwardly rectifying potassium channels. The current density recorded was low (0.5+/-0.1 pA pF(-1), n = 8). These results indicate that, in two different vascular preparations, barium sensitive-inwardly rectifying potassium conductance and the ouabain sensitive-Na+/K+ pump are not involved in the EDHF-mediated hyperpolarization. Furthermore, potassium did not mimic the effect of EDHF pointing out that potassium and EDHF are not the same entity in those arteries.

A novel tetrodotoxin-sensitive Na+ current in cultured human coronary myocytes.

Voltage-gated Na+ currents (INaS) are usually not found in arterial smooth muscle. We enzymatically isolated myocytes from the media of left coronary arteries of heart transplant patients with ischemic cardiopathy. Using the whole-cell voltage-clamp technique (20 degrees C to 22 degrees C), we detected no INa in any of the freshly isolated myocytes. In contrast, when the cells were grown in culture, we could record a large INa. This INa was characterized by a biexponential decay comprising a fast inactivating and sustained components that could not be separated by their electrophysiological and pharmacological properties. INa activated at depolarizations positive to -50 mV, was maximal at 0 mV, and was available from relatively low resting membrane potentials (half-inactivation at -46 mV). INa was modulated by several ligands known to bind selectively at different sites of Na+ channels. It was blocked with high affinity by tetrodotoxin (IC50, approximately 10 nmol/L) and local anesthetics (bupivacaine and lidocaine; IC50, approximately 100 nmol/L) and by Cd2+ (IC50, approximately 300 mumol/L). INa was modulated by Na+ channel agonists such as toxin AsV from Anemonia sulcata and veratridine, which slowed current kinetics dramatically. In conclusion, human coronary myocytes in culture can express an atypical tetrodotoxin-sensitive INa with a large sustained component, which is expected to contribute to massive Na+ influx into these cells. Phenotypic modulation of the expression of this INa may be related to cell dedifferentiation and proliferation.

Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide.

Voltage-gated Ca2+ channels contribute to the maintenance of contractile tone in vascular myocytes and are potential targets for vasodilating agents. There is no information available about their nature and regulation in human coronary arteries. We used the whole-cell voltage-clamp technique to characterize Ca2+-channel currents immediately after enzymatic dissociation and after primary culture of coronary myocytes taken from heart transplant patients. We recorded a dihydropyridine-sensitive L-type current in both freshly isolated and primary cultured cells. A T-type current was recorded only in culture. The L- (but not the T-) type current was inhibited by permeable analogues of cGMP in a dose-dependent manner. This effect was mimicked by the nitric oxide-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine which increased intracellular cGMP. Methylene blue, known to inhibit guanylate cyclase, antagonized the effect of SNAP. Inhibitions by SNAP and cGMP were not additive and seemed to occur through a common pathway. We conclude that (a) L-type Ca2+ channels are the major pathway for voltage-gated Ca2+ entry in human coronary myocytes; (b) their inhibition by agents stimulating nitric oxide and/or intracellular cGMP production is expected to contribute to vasorelaxation and may be involved in the therapeutic effect of nitrovasodilators; and (c) the expression of T-type Ca2+ channels in culture may be triggered by cell proliferation.

Absence of calcium channels in neonatal rat aortic myocytes.

We have investigated whole-cell Ba2+ currents through Ca2+ channels (IBa) in single myocytes freshly isolated from the aortic media of neonatal (1-day-old) and adult (12-week-old) rats. In neonatal myocytes, (IBa) was undetectable even in presence of the dihydropyridine (DHP) agonist Bay K 8644. Binding of [3H]Nitrendipine on crude plasma membrane preparation of media confirmed the absence of DHP-receptors. By contrast, a robust DHP-sensitive 'L-type' IBa was recorded in adults which was consistent with the presence of specific [3H]Nitrendipine binding sites. In conclusion, neonatal aortic myocytes do not express any Ca2+ channels. The acquisition of L-type Ca2+ channels may be related to cell differentiation and acquisition of contractility during postnatal development.

Blockade of nitric oxide synthesis by tyrosine kinase inhibitors in neurones.

In striatal neurones in culture, N-methyl-D-aspartate-(NMDA), kainate-(Kai) and K(+)-dependent cGMP production is entirely mediated via nitric oxide (NO). Low concentrations of lavendustin-A (< or = 0.3 microM), a highly specific tyrosine kinase inhibitor, reduced irreversibly and in a time-dependent manner NMDA-stimulated cGMP production. After a preincubation period of 20 min with lavendustin-A (0.3 microM), the inhibition of NMDA-induced cGMP production was equal to 56 +/- 8% (n = 6). After the same preincubation period, the IC50 of the lavendustin-A blockade was 30 +/- 15 nM. Genistein, another tyrosine kinase inhibitor also inhibited NMDA-dependent cGMP production with high potencies (< or = 3 microM). Whatever the tyrosine kinase inhibitor tested, the basal cGMP production remained unaffected. Kai-, K(+)-, and ionomycin-induced cGMP production was also inhibited by lavendustin-A, and genistein. In contrast, tyrosine kinase inhibitors were unable to block NO donor-induced cGMP production. Using patch clamp experiments, we have also found that lavendustin-A (0.3-1 microM), the most potent tyrosine kinase inhibitor used, (a) did not reduce the NMDA receptor-mediated current, (b) only slighly affected Kai receptor-mediated current (16.4 +/- 3.4% inhibition) and (c) had a marked effect on voltage-sensitive Ca2+ channel- (VSCC) mediated currents (44.4 +/- 4.9% inhibition). A reduction in VSCC activity certainly explains the inhibition of K(+)-, Kai- and possibly part of the NMDA-induced cGMP production.(ABSTRACT TRUNCATED AT 250 WORDS)