Jean-François Quignard

Subunit‐specific modulation of T‐type calcium channels by zinc

Zinc (Zn2+) functions as a signalling molecule in the nervous system and modulates many ionic channels. In this study, we have explored the effects of Zn2+ on recombinant T‐type calcium channels (CaV3.1, CaV3.2 and CaV3.3). Using tsA‐201 cells, we demonstrate that CaV3.2 current (IC50, 0.8 μm) is significantly more sensitive to Zn2+ than are CaV3.1 and CaV3.3 currents (IC50, 80 μm and ∼160 μm, respectively). This inhibition of CaV3 currents is associated with a shift to more negative membrane potentials of both steady‐state inactivation for CaV3.1, CaV3.2 and CaV3.3 and steady‐state activation for CaV3.1 and CaV3.3 currents. We also document changes in kinetics, especially a significant slowing of the inactivation kinetics for CaV3.1 and CaV3.3, but not for CaV3.2 currents. Notably, deactivation kinetics are significantly slowed for CaV3.3 current (∼100‐fold), but not for CaV3.1 and CaV3.2 currents. Consequently, application of Zn2+ results in a significant increase in CaV3.3 current in action potential clamp experiments, while CaV3.1 and CaV3.2 currents are significantly reduced. In neuroblastoma NG 108‐15 cells, the duration of CaV3.3‐mediated action potentials is increased upon Zn2+ application, indicating further that Zn2+ behaves as a CaV3.3 channel opener. These results demonstrate that Zn2+ exhibits differential modulatory effects on T‐type calcium channels, which may partly explain the complex features of Zn2+ modulation of the neuronal excitability in normal and disease states.

T‐type Cav3.3 calcium channels produce spontaneous low‐threshold action potentials and intracellular calcium oscillations

The precise contribution of T‐type Ca2+ channels in generating action potentials (APs), burst firing and intracellular Ca2+ signals needs further elucidation. Here, we show that Cav3.3 channels can trigger repetitive APs, generating spontaneous membrane potential oscillations (MPOs), and a concomitant increase in the intracellular Ca2+ concentration ([Ca2+]i) when overexpressed in NG108‐15 cells. MPOs were dependent on Cav3.3 channel activity given that they were recorded from a potential range of −55 to −70 mV, blocked by nickel and mibefradil, as well as by low external Ca2+ concentration. APs of distinct duration were recorded: short APs (sAP) or prolonged APs (pAP) with a plateau potential near −40 mV. The voltage‐dependent properties of the Cav3.3 channels constrained the AP duration and the plateau potential was supported by sustained calcium current through Cav3.3 channels. The sustained current amplitude decreased when the resting holding potential was depolarized, thereby inducing a switch of AP shape from pAP to sAP. Duration of the [Ca2+]i oscillations was also closely related to the shape of APs. The Cav3.3 window current was the oscillation trigger as shown by shifting the Cav3.3 window current potential range as a result of increasing the external Ca2+ concentration, which resulted in a corresponding shift of the AP threshold. Overall, the data demonstrate that the Cav3.3 window current is critical in triggering intrinsic electrical and [Ca2+]i oscillations. The functional expression of Cav3.3 channels can generate spontaneous low‐threshold calcium APs through its window current, indicating that Cav3.3 channels can play a primary role in pacemaker activity.

Stretch-induced Ca2+ signalling in vascular smooth muscle cells depends on Ca2+ store segregation

AIM Calcium is a key second messenger that can be mobilized from both the extracellular medium and intracellular calcium stores. Pulmonary arterial smooth muscle cells (PASMCs) respond to stretch by a calcium increase, a mechanism enhanced during pulmonary hypertension (PH). We investigated the role of the spatial organization between plasma membrane stretch-activated channels (SACs) and intracellular calcium stores [sarcoplasmic reticulum (SR), mitochondria, and lysosomes) in response to stretch. METHODS AND RESULTS Studies were performed in freshly isolated PASMCs from both control and two different rat models of PH (chronically hypoxic and monocrotaline-treated rats). Co-immunolabellings revealed that the subcellular segregation between each subtype of SR ryanodine receptors (RyR1, RyR2, and RyR3), SERCA2 pumps (SERCA2a and SERCA2b), mitochondria, or lysosomes in freshly isolated PASMCs differs from control and PH PASMCs. In control PASMCs, stretching the membrane activates a Ca(2+) influx through SACs. This influx is amplified by cell hyperpolarization, a calcium release by subplasmalemmal RyR1 and is then buffered by mitochondria. In two different PH rat models, the calcium response to stretch is enhanced due to hyper-reactivity of SACs and a greater calcium amplification by all RyR subtypes. CONCLUSION The spatial organization of RyR and calcium stores in PASMCs is important for cell signalling and plays a causal role in PH.

Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats.

There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca(2+)](i)). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca(2+)](i) increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca(2+) release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.

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 μm) or 10 μm InsP6 had no action on Ba2+ current, whereas 50 μm InsP6 or 10 μm InsP5 plus 10 μm 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 μm) or calphostin C (100 nm) abolished the InsP5,6‐induced stimulation. Neither the PKA inhibitor H89 (1 μm) nor the protein phosphatase inhibitors okadaic acid (500 nm) or cypermethrin (1 μm) 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.

Beneficial effect of dehydroepiandrosterone on pulmonary hypertension in a rodent model of pulmonary hypertension in infants.

Background:Pulmonary hypertension (PH) is a disease that affects the adult or infant population. Dehydroepiandrosterone (DHEA), a steroid hormone, has been previously shown to prevent and to reverse PH in an adult rat model. We thus investigated its effect in a rat-pup model of chronic hypoxic PH.Methods:Animals were maintained for 3 wk in a hypobaric chamber to induce PH, with or without concomitant treatment with DHEA (30 mg/kg every alternate day).Results:DHEA significantly reduced mean pulmonary artery pressure (measured by right cardiac catheterization), pulmonary artery remodeling (evaluated by histology), and right-ventricular hypertrophy (measured by echography and by the Fulton index). At the level of the pulmonary artery smooth muscle cell (PASMC), DHEA increased activity and expression of the large-conductance Ca2+-activated potassium channel (BKCa) (assessed by means of the patch clamp technique). DHEA also inhibited both serotonin- and KCl-induced contraction and smooth muscle cell proliferation.Conclusion:Collectively, these results indicate that DHEA prevents PH in infant rats and may therefore be clinically relevant for the management of PH in human infants.

Caveolae are involved in mechanotransduction during pulmonary hypertension

Caveolae are stiff plasma membrane microdomains implicated in various cell response mechanisms like Ca(2+) signaling and mechanotransduction. Pulmonary arterial smooth muscle cells (PASMC) transduce mechanical stimuli into Ca(2+) increase via plasma membrane stretch-activated channels (SAC). This mechanotransduction process is modified in pulmonary hypertension (PH) during which stretch forces are increased by the increase in arterial blood pressure. We propose to investigate how caveolae are involved in the pathophysiology of PH and particularly in mechanotransduction. PASMC were freshly isolated from control rats (Ctrl rats) and rats suffering from PH induced by 3 wk of chronic hypoxia (CH rats). Using a caveolae disrupter (methyl-β-cyclodextrin), we showed that SAC activity measured by patch-clamp, stretch-induced Ca(2+) increase measured with indo-1 probe and pulmonary arterial ring contraction to osmotic shock are enhanced in Ctrl rats when caveolae are disrupted. In CH rats, SAC activity, Ca(2+), and contraction responses to stretch are all higher compared with Ctrl rats. However, in contrast to Ctrl rats, caveolae disruption in CH-PASMC, reduces SAC activity, Ca(2+) responses to stretch and arterial contractions. Furthermore, by means of immunostainings and transmission electron microscopy, we observed that caveolae and caveolin-1 are expressed in PASMC from both Ctrl and CH rats and localize close to subplasmalemmal sarcoplasmic reticulum (ryanodine receptors) and mitochondria, thus facilitating Ca(2+) exchanges, particularly in CH. In conclusion, caveolae are implicated in mechanotransduction in Ctrl PASMC by buffering mechanical forces. In PH-PASMC, caveolae form a distinct Ca(2+) store facilitating Ca(2+) coupling between SAC and sarcoplasmic reticulum.

Altered vasoreactivity in neonatal rats with pulmonary hypertension associated with bronchopulmonary dysplasia: Implication of both eNOS phosphorylation and calcium signaling.

Bronchopulmonary dysplasia (BPD) consists of an arrest of pulmonary vascular and alveolar growth, with persistent hypoplasia of the pulmonary microvasculature and alveolar simplification. In 25 to 40% of the cases, BPD is complicated by pulmonary hypertension (BPD-PH) that significantly increases the risk of morbidity. In vivo studies suggest that increased pulmonary vascular tone could contribute to late PH in BPD. Nevertheless, an alteration in vasoreactivity as well as the mechanisms involved remain to be confirmed. The purpose of this study was thus to assess changes in pulmonary vascular reactivity in a murine model of BPD-PH. Newborn Wistar rats were exposed to either room air (normoxia) or 90% O2 (hyperoxia) for 14 days. Exposure to hyperoxia induced the well-known features of BPD-PH such as elevated right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and decreased pulmonary vascular density. Intrapulmonary arteries from hyperoxic pups showed decreased endothelium-dependent relaxation to acetylcholine without any alteration of relaxation to the NO-donor sodium nitroprusside. This functional alteration was associated with a decrease of lung eNOS phosphorylation at the Ser1177 activating site. In pups exposed to hyperoxia, serotonin and phenylephrine induced exacerbated contractile responses of intrapulmonary arteries as well as intracellular calcium response in pulmonary arterial smooth muscle cells (PASMC). Moreover, the amplitude of the store-operated Ca2+ entry (SOCE), induced by store depletion using a SERCA inhibitor, was significantly greater in PASMC from hyperoxic pups. Altogether, hyperoxia-induced BPD-PH alters the pulmonary arterial reactivity, with effects on both endothelial and smooth muscle functions. Reduced activating eNOS phosphorylation and enhanced Ca2+ signaling likely account for alterations of pulmonary arterial reactivity.

Calcium signalling induced by in vitro exposure to silicium dioxide nanoparticles in rat pulmonary artery smooth muscle cells

The development and use of nanomaterials, especially engineered nanoparticles (NP), is expected to provide many benefits. But at the same time the development of such materials is also feared because of their potential human health risks. Indeed, NP display some characteristics similar to ultrafine environmental particles which are known to exert deleterious cardiovascular effects including pro-hypertensive ones. In this context, the effect of NP on calcium signalling, whose deregulation is often involved in hypertensive diseases, remain poorly described. We thus assessed the effect of SiO2 NP on calcium signalling by fluorescence imaging and on the proliferation response in rat pulmonary artery smooth muscle cells (PASMC). In PASMC, acute exposure to SiO2 NP, from 1 to 500μg/mL, produced an increase of the [Ca2+]i. In addition, when PASMC were exposed to NP at 200μg/mL, a proliferative response was observed. This calcium increase was even greater in PASMC isolated from rats suffering from pulmonary hypertension. The absence of extracellular calcium, addition of diltiazem or nicardipine (L-type voltage-operated calcium channel inhibitors both used at 10μM), and addition of capsazepine or HC067047 (TRPV1 and TRPV4 inhibitors used at 10μM and 5μM, respectively) significantly reduced this response. Moreover, this response was also inhibited by thapsigargin (SERCA inhibitor, 1μM), ryanodine (100μM) and dantrolene (ryanodine receptor antagonists, 10μM) but not by xestospongin C (IP3 receptor antagonist, 10μM). Thus, NP induce an intracellular calcium rise in rat PASMC originating from both extracellular and intracellular calcium sources. This study also provides evidence for the implication of TRPV channels in NP induced calcium rise that may highlight the role of these channels in the deleterious cardiovascular effects of NP.