Multidrug resistance-associated protein 4 (MRP4, also known as Abcc4) regulates intracellular levels of cAMP and cGMP in arterial SMCs. Here, we report our studies of the role of MRP4 in the development and progression of pulmonary arterial hypertension (PAH), a severe vascular disease characterized by chronically elevated pulmonary artery pressure and accompanied by remodeling of the small pulmonary arteries as a prelude to right heart failure and premature death. MRP4 expression was increased in pulmonary arteries from patients with idiopathic PAH as well as in WT mice exposed to hypoxic conditions. Consistent with a pathogenic role for MRP4 in PAH, WT mice exposed to hypoxia for 3 weeks showed reversal of hypoxic pulmonary hypertension (PH) following oral administration of the MRP4 inhibitor MK571, and Mrp4 -/-mice were protected from hypoxic PH. Inhibition of MRP4 in vitro was accompanied by increased intracellular cAMP and cGMP levels and PKA and PKG activities, implicating cyclic nucleotide-related signaling pathways in the mechanism underlying the protective effects of MRP4 inhibition. Our data suggest that MRP4 could represent a potential target for therapeutic intervention in PAH.

BackgroundEndothelial control of vascular smooth muscle plays a major role in the resulting vasoreactivity implicated in physiological or pathological circulatory processes. However, a comprehensive understanding of endothelial (EC)/smooth muscle cells (SMC) crosstalk is far from complete. Here, we have examined the role of gap junctions and reactive oxygen species (ROS) in this crosstalk and we demonstrate an active contribution of SMC to endothelial control of vasomotor tone.Methodology/Principal FindingsIn small intrapulmonary arteries, quantitative RT-PCR, Western Blot analyses and immunofluorescent labeling evidenced connexin (Cx) 37, 40 and 43 in EC and/or SMC. Functional experiments showed that the Cx-mimetic peptide targeted against Cx 37 and Cx 43 (37,43Gap27) (1) reduced contractile and calcium responses to serotonin (5-HT) simultaneously recorded in pulmonary arteries and (2) abolished the diffusion in SMC of carboxyfluorescein-AM loaded in EC. Similarly, contractile and calcium responses to 5-HT were decreased by superoxide dismutase and catalase which, catabolise superoxide anion and H2O2, respectively. Both Cx- and ROS-mediated effects on the responses to 5-HT were reversed by L-NAME, a NO synthase inhibitor or endothelium removal. Electronic paramagnetic resonance directly demonstrated that 5-HT-induced superoxide anion production originated from the SMC. Finally, whereas 5-HT increased NO production, it also decreased cyclic GMP content in isolated intact arteries.Conclusions/SignificanceThese data demonstrate that agonist-induced ROS production in SMC targeting EC via myoendothelial gap junctions reduces endothelial NO-dependent control of pulmonary vasoreactivity. Such SMC modulation of endothelial control may represent a signaling pathway controlling vasoreactivity under not only physiological but also pathological conditions that often implicate excessive ROS production.

These data provide evidence that hypoxic circulating MPs induce endothelial dysfunction in rat aorta and pulmonary arteries by decreasing NO production. Moreover, MPs display tissue specificity with respect to increased oxidative stress, which occurs only in pulmonary ECs.

5-Hydroxytryptamine (5-HT) is a potent pulmonary vasoconstrictor and contributes to hypoxic pulmonary vasoconstriction and pulmonary arterial hypertension. Small intrapulmonary vessels are very sensitive to hypoxia and play a major role for blood flow regulation in the lung. Thus we have investigated the mechanisms involved in the calcium signal to 5-HT in rat small intrapulmonary artery (IPA). Effects of 5-HT were examined in isolated IPA (external diameter <250 microm) from rat. Digital imaging with fura-PE3 was used to record intracellular calcium concentration ([Ca(2+)](i)) and to follow external diameter of the vessels. 5-HT induced a sustained [Ca(2+)](i) variation that was sensitive to the inhibitor of the 5-HT(2A) receptors, ketanserin, and insensitive to voltage-dependent L-type calcium channel blockers (nitrendipine and nicardipine) or voltage-independent calcium channel antagonists (LOE-908, SKF-96365, and gadolinium). The calcium response to 5-HT was also not modified by a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor (cyclopiazonic acid; CPA), which depletes intracellular calcium stores. CPA alone activated a capacitative calcium channel that was sensitive to LOE-908 and insensitive to SKF-96365 and gadolinium. The sustained calcium signal to 5-HT was partly blocked by inhibitors of arachidonic acid production (RHC-80267 and isotetrandrine) and mimicked by application of exogenous arachidonic acid. These results suggest that activation of a noncapacitative, arachidonic acid-sensitive, receptor-operated calcium channel contributes to 5-HT-induced sustained calcium increase in small IPA.

Background: The airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR) expressed in airway epithelial cells.

The effect of endothelin (ET)-1 on both cytosolic Ca2+ concentration ([Ca2+]i) and membrane current in freshly isolated myocytes, as well as on the contraction of arterial rings, was investigated in rat main pulmonary artery (RMPA) and intrapulmonary arteries (RIPA). ET-1 (5–100 nM, 30 s) induced a first [Ca2+]ipeak followed by 3–5 oscillations of decreasing amplitude. In RMPA, the ET-1-induced [Ca2+]iresponse was fully abolished by BQ-123 (0.1 μM). In RIPA, the response was inhibited by BQ-123 in only 21% of the cells, whereas it was abolished by BQ-788 (1 μM) in 70% of the cells. In both types of arteries, the response was not modified in the presence of 100 μM La3+ or in the absence of external Ca2+ but disappeared after pretreatment of the cells with thapsigargin (1 μM) or neomycin (0.1 μM). In RPMA myocytes clamped at −60 mV, ET-1 induced an oscillatory inward current, the reversal potential of which was close to the equilibrium potential for Cl−. This current was unaltered by the removal of external Ca2+ but was abolished by niflumic acid (50 μM). In arterial rings, the ET-1 (100 nM)-induced contraction was decreased by 35% in the presence of either niflumic acid (50 μM) or nifedipine (1 μM). These results demonstrate that ET-1 via the ETA receptor only in RMPA and both ETA and ETB receptors in RIPA induce [Ca2+]ioscillations due to iterative Ca2+release from an inositol trisphosphate-sensitive Ca2+ store. Ca2+ release secondarily activates an oscillatory membrane Cl−current that can depolarize the cell membrane, leading to an influx of Ca2+, this latter contributing to the ET-1-induced vasoconstrictor effect.

BackgroundPulmonary hypertension (PH) is characterized by arterial vascular remodelling and alteration in vascular reactivity. Since gap junctions are formed with proteins named connexins (Cx) and contribute to vasoreactivity, we investigated both expression and role of Cx in the pulmonary arterial vasoreactivity in two rat models of PH.MethodsIntrapulmonary arteries (IPA) were isolated from normoxic rats (N), rats exposed to chronic hypoxia (CH) or treated with monocrotaline (MCT). RT-PCR, Western Blot and immunofluorescent labelling were used to study the Cx expression. The role of Cx in arterial reactivity was assessed by using isometric contraction and specific gap junction blockers. Contractile responses were induced by agonists already known to be involved in PH, namely serotonin, endothelin-1 and phenylephrine.ResultsCx 37, 40 and 43 were expressed in all rat models and Cx43 was increased in CH rats. In IPA from N rats only, the contraction to serotonin was decreased after treatment with 37-43Gap27, a specific Cx-mimetic peptide blocker of Cx 37 and 43. The contraction to endothelin-1 was unchanged after incubation with 40Gap27 (a specific blocker of Cx 40) or 37-43Gap27 in N, CH and MCT rats. In contrast, the contraction to phenylephrine was decreased by 40Gap27 or 37-43Gap27 in CH and MCT rats. Moreover, the contractile sensitivity to high potassium solutions was increased in CH rats and this hypersensitivity was reversed following 37-43Gap27 incubation.ConclusionAltogether, Cx 37, 40 and 43 are differently expressed and involved in the vasoreactivity to various stimuli in IPA from different rat models. These data may help to understand alterations of pulmonary arterial reactivity observed in PH and to improve the development of innovative therapies according to PH aetiology.

1 Cytosolic calcium concentration ([Ca 2+ ] i ) by indo 1 microspectro¯uorimetry 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 mM) caused a rapid and graded increase in [Ca 2+ ] i up to a net amplitude of 492+26 nM (n=19) which gradually declined. The EC 50 for ACh was 0.13 mM. This ®rst [Ca ] i response to ACh but with a higher IC 50 of 2 mM. Methoctramine (a selective M 2 antagonist) up to a concentration of 10 mM caused only a 40% inhibition. The eect 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 mM, and methoctramine at 1, 3 and 10 mM. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA 2 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 [Ca 2+ ] i oscillations. The occurrence of these oscillations depends on the graded amplitude of the ®rst [Ca 2+ ] i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca 2+ ] i and the contractile responses are primarily dependent on activation of the M 3 receptor subtype.