Leptin Induces Epigenetic Regulation of Transient Receptor Potential Melastatin 7 in Rat Adrenal Pheochromocytoma Cells.

Obesity elevates the plasma level of leptin, which has been associated with hypertension. Our recent studies in mice demonstrated that leptin increases blood pressure by activating the carotid sinus nerve, which transmits the chemosensory input from carotid bodies (CBs) to the medullary centers, and that the effect of leptin is mediated via Trpm7 (TRP [transient receptor potential] melastatin 7) channels in CB glomus cells. We also found that Trpm7 overexpression and Trpm7 promoter demethylation in CBs correlate positively with the hyperleptinemia and leptin receptor overexpression in CBs. Hence, we postulated that leptin epigenetically regulates Trpm7 expression in CBs. We addressed our hypothesis by using rat adrenal pheochromocytoma (PC12) cells as a model of CB glomus cells. PC12 cells expressing LEPRb (long, active form of leptin receptor) showed dramatic induction of the promoter activity and expression of Trpm7 upon leptin treatment. The increased Trpm7 expression coincided with the reduction of CpG site-specific methylation and trimethylation of H3K27 (H3 [histone 3] K27 [lysine 27]) and the increase of acetylation of H3K27 and trimethylation of H3K4 (H3 lysine 4) at the Trpm7 promoter. The inhibitor of STAT3 (signal transducer and activator of transcription 3) signaling, SD1008, reversed the leptin-induced Trpm7 promoter activity via modulations of the binding of pSTAT3 (phosphorylated STAT3) and DNMT3B (DNA methyltransferase 3B) and modifications of H3K27 and H3K4 at the Trpm7 promoter. Our results suggest that leptin-activated pSTAT3 epigenetically regulates the transcription of Trpm7 through DNA methylation and histone modifications. Because epigenetic changes are reversible, targeting epigenetic modifications of Trpm7 may serve as a new therapeutic approach for the treatment of hypertension in obesity.

Increased intracellular Cl- concentration in pulmonary arterial myocytes is associated with chronic hypoxic pulmonary hypertension.

Chloride channels play an important role in regulating smooth muscle contraction and proliferation, and contribute to the enhanced constriction of pulmonary arteries (PAs) in pulmonary hypertension (PH). The intracellular Cl- concentration ([Cl-]i), tightly regulated by various Cl- transporters, determines the driving force for Cl- conductance, thereby the functional outcome of Cl- channel activation. This study characterizes for the first time the expression profile of Cl- transporters/exchangers in PA smooth muscle and provides the first evidence that the intracellular Cl- homeostasis is altered in PA smooth muscle cells (PASMCs) associated with chronic hypoxic PH (CHPH). Quantitative RT-PCR revealed that the endothelium-denuded intralobar PA of rats expressed Slc12a gene family-encoded Na-K-2Cl cotransporter 1 (NKCC1), K-Cl cotransporters (KCC) 1, 3, and 4, and Slc4a gene family-encoded Na+-independent and Na+-dependent Cl-/HCO3- exchangers. Exposure of rats to chronic hypoxia (10% O2, 3 wk) caused CHPH and selectively increased the expression of Cl--accumulating NKCC1 and reduced the Cl--extruding KCC4. The intracellular Cl- concentration ([Cl-]i) averaged at 45 mM and 47 mM in normoxic PASMCs as determined by fluorescent indicator MEQ and by gramicidin-perforated patch-clamp technique, respectively. The ([Cl-]i was increased by ∼10 mM in PASMCs of rats with CHPH. Future studies are warranted to further establish the hypothesis that the altered intracellular Cl- homeostasis contributes to the pathogenesis of CHPH.

Leptin Induces Hypertension Acting on Transient Receptor Potential Melastatin 7 Channel in the Carotid Body.

RATIONALE: Obesity leads to resistant hypertension and mechanisms are poorly understood, but high plasma levels of leptin have been implicated. Leptin increases blood pressure acting both centrally in the dorsomedial hypothalamus and peripherally. Sites of the peripheral hypertensive effect of leptin have not been identified. We previously reported that leptin enhanced activity of the carotid sinus nerve, which transmits chemosensory input from the carotid bodies (CBs) to the medullary centers, and this effect was abolished by nonselective blockers of Trp (transient receptor potential) channels. We searched our mouse CB transcriptome database and found that the Trpm7 (transient receptor potential melastatin 7) channel was the most abundant Trp channel. OBJECTIVE: To examine if leptin induces hypertension acting on the CB Trpm7. METHODS AND RESULTS: C57BL/6J (n=79), leptin receptor (LepRb) deficient db/db mice (n=22), and LepRb-EGFP (n=4) mice were used. CB Trpm7 and LepRb gene expression was determined and immunohistochemistry was performed; CB glomus cells were isolated and Trpm7-like current was recorded. Blood pressure was recorded continuously in (1) leptin-treated C57BL/6J mice with intact and denervated CB; (2) leptin-treated C57BL/6J mice, which also received a nonselective Trpm7 blocker FTY720 administered systemically or topically to the CB area; (3) leptin-treated C57BL/6J mice transfected with Trpm7 small hairpin RNA to the CB, and (4) Leprb deficient obese db/db mice before and after Leprb expression in CB. Leptin receptor and Trpm7 colocalized in the CB glomus cells. Leptin induced a nonselective cation current in these cells, which was inhibited by Trpm7 blockers. Leptin induced hypertension in C57BL/6J mice, which was abolished by CB denervation, Trpm 7 blockers, and Trpm7 small hairpin RNA applied to CBs. Leprb overexpression in CB of Leprb-deficient db/db mice demethylated the Trpm7 promoter, increased Trpm7 gene expression, and induced hypertension. CONCLUSIONS: We conclude that leptin induces hypertension acting on Trmp7 in CB, which opens horizons for new therapy.

CD38 mediates angiotensin II-induced intracellular Ca(2+) release in rat pulmonary arterial smooth muscle cells.

CD38 is a multifunctional enzyme that catalyzes the formation of the endogenous Ca(2+)-mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors (RyRs) of sarcoplasmic reticulum and NAADP-sensitive Ca(2+) release channels in endolysosomes, respectively. It plays important roles in systemic vascular functions, but there is little information on CD38 in pulmonary arterial smooth muscle cells (PASMCs). Earlier studies suggested a redox-sensing role of CD38 in hypoxic pulmonary vasoconstriction. This study sought to characterize its roles in angiotensin II (Ang II)-induced Ca(2+) release (AICR) in PASMCs. Examination of CD38 expression in various rat arteries found high levels of CD38 mRNA and protein in pulmonary arteries. The Ang II-elicited Ca(2+) response consisted of extracellular Ca(2+) influx and intracellular Ca(2+) release in PASMCs. AICR activated in the absence of extracellular Ca(2+) was reduced by pharmacological or siRNA inhibition of CD38, by the cADPR antagonist 8-bromo-cADPR or ryanodine, and by the NAADP antagonist Ned-19 or disruption of endolysosomal Ca(2+) stores with the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Suppression of AICR by the inhibitions of cADPR- and NAADP-dependent pathways were nonadditive, indicating interdependence of RyR- and NAADP-gated Ca(2+) release. Furthermore, AICR was inhibited by the protein kinase C inhibitor staurosporine, the nonspecific NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium, the NOX2-specific inhibitor gp91ds-tat, and the scavenger of reactive oxygen species (ROS) tempol. These results provide the first evidence that Ang II activates CD38-dependent Ca(2+) release via the NOX2-ROS pathway in PASMCs.

Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells.

BACKGROUND/AIMS: Adenosine diphosphate ribose (ADPR), a product of ß-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. METHODS: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. RESULTS: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. CONCLUSION: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells.

Intracellular calcium (Ca(2+)) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca(2+) signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca(2+) is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca(2+) is crucial for excitation-transcription coupling. However, information on Ca(2+) signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca(2+) signals, genetically encoded Ca(2+) indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca(2+) signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca(2+) of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin-permeabilized cells with 1,4,5-trisphosphate (IP3) elicited a transient elevation of Ca(2+) concentration with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G protein-coupled receptors by endothelin-1 and angiotensin II preferentially elevated the subsarcolemmal Ca(2+) signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet-derived growth factor, elicited Ca(2+) signals with similar amplitudes in all three regions, except that the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca(2+) signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca(2+) signals in PASMCs.

Nicotinic acid adenine dinucleotide phosphate (NAADP) activates global and heterogeneous local Ca2+ signals from NAADP- and ryanodine receptor-gated Ca2+ stores in pulmonary arterial myocytes.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing messenger that releases Ca(2+) from endolysosomal organelles. Recent studies showed that NAADP-induced Ca(2+) release is mediated by the two-pore channels (TPCs) TPC1 and TPC2. However, the expression of TPCs and the NAADP-induced local Ca(2+) signals have not been examined in vascular smooth muscle. Here, we found that both TPC1 and TPC2 are expressed in rat pulmonary arterial smooth muscle cells (PASMCs), with TPC1 being the major subtype. Application of membrane-permeant NAADP acetoxymethyl ester to PASMCs elicited a biphasic increase in global [Ca(2+)]i, which was independent of extracellular Ca(2+) and blocked by the NAADP antagonist Ned-19 or the vacuolar H(+)-ATPase inhibitor bafilomycin A1, indicating Ca(2+) release from acidic endolysosomal Ca(2+) stores. The Ca(2+) response was unaffected by xestospongin C but was partially blocked by ryanodine or thapsigargin. NAADP triggered heterogeneous local Ca(2+) signals, including a diffuse increase in cytosolic [Ca(2+)], Ca(2+) sparks, Ca(2+) bursts, and regenerative Ca(2+) release. The diffuse Ca(2+) increase and Ca(2+) bursts were ryanodine-insensitive, presumably arising from different endolysosomal sources. Ca(2+) sparks and regenerative Ca(2+) release were inhibited by ryanodine, consistent with cross-activation of loosely coupled ryanodine receptors. Moreover, Ca(2+) release stimulated by endothelin-1 was inhibited by Ned-19, ryanodine, or xestospongin C, suggesting that NAADP-mediated Ca(2+) signals interact with both ryanodine and inositol 1,4,5-trisphosphate receptors during agonist stimulation. Our results show that NAADP mediates complex global and local Ca(2+) signals. Depending on the physiological stimuli, these diverse Ca(2+) signals may serve to regulate different cellular functions in PASMCs.

TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension.

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive channel in pulmonary arterial smooth muscle cells (PASMCs). Its upregulation by chronic hypoxia is associated with enhanced myogenic tone, and genetic deletion of trpv4 suppresses the development of chronic hypoxic pulmonary hypertension (CHPH). Here we further examine the roles of TRPV4 in agonist-induced pulmonary vasoconstriction and in the enhanced vasoreactivity in CHPH. Initial evaluation of TRPV4-selective antagonists HC-067047 and RN-1734 in KCl-contracted pulmonary arteries (PAs) of trpv4(-/-) mice found that submicromolar HC-067047 was devoid of off-target effect on pulmonary vasoconstriction. Inhibition of TRPV4 with 0.5 µM HC-067047 significantly reduced the sensitivity of serotonin (5-HT)-induced contraction in wild-type (WT) PAs but had no effect on endothelin-1 or phenylephrine-activated response. Similar shift in the concentration-response curve of 5-HT was observed in trpv4(-/-) PAs, confirming specific TRPV4 contribution to 5-HT-induced vasoconstriction. 5-HT-induced Ca(2+) response was attenuated by HC-067047 in WT PASMCs but not in trpv4(-/-) PASMCs, suggesting TRPV4 is a major Ca(2+) pathway for 5-HT-induced Ca(2+) mobilization. Nifedipine also attenuated 5-HT-induced Ca(2+) response in WT PASMCs but did not cause further reduction in the presence of HC-067047, suggesting interdependence of TRPV4 and voltage-gated Ca(2+) channels in the 5-HT response. Chronic exposure (3-4 wk) of WT mice to 10% O2 caused significant increase in 5-HT-induced maximal contraction, which was partially reversed by HC-067047. In concordance, the enhancement of 5-HT-induced contraction was significantly reduced in PAs of CH trpv4(-/-) mice and HC-067047 had no further effect on the 5-HT induced response. These results suggest unequivocally that TRPV4 contributes to 5-HT-dependent pharmaco-mechanical coupling and plays a major role in the enhanced pulmonary vasoreactivity to 5-HT in CHPH.

Chronic hypoxia-induced upregulation of Ca2+-activated Cl- channel in pulmonary arterial myocytes: a mechanism contributing to enhanced vasoreactivity.

Chronic hypoxic pulmonary hypertension (CHPH) is associated with altered expression and function of cation channels in pulmonary arterial smooth muscle cells (PASMCs), but little is known for anion channels. The Ca(2+)-activated Cl(-) channel (CaCC), recently identified as TMEM16A, plays important roles in pulmonary vascular function. The present study sought to determine the effects of chronic hypoxia (CH) on the expression and function of CaCCs in PASMCs, and their contributions to the vascular hyperreactivity in CHPH. Male Wistar rats were exposed to room air or 10% O(2) for 3­4 weeks to generate CHPH. CaCC current (I(CI.Ca)) elicited by caffeine-induced Ca(2+) release or by depolarization at a constant high [Ca(2+)](i) (500 or 750 nm) was significantly larger in PASMCs of CH rats compared to controls. The enhanced I(CI.Ca)) density in CH PASMCs was unrelated to changes in amplitude of Ca(2+) release, Ca(2+)-dependent activation, voltage-dependent properties or calcineurin-dependent modulation of CaCCs, but was associated with increased TMEM16A mRNA and protein expression. Maximal contraction induced by serotonin, an important mediator of CHPH, was potentiated in endothelium-denuded pulmonary arteries of CH rats. The enhanced contractile response was prevented by the CaCC blockers niflumic acid and T16A(inh)-A01, or by the L-type Ca(2+) channel antagonist nifedipine. The effects of niflumic acid and nifedipine were non-additive. Our results demonstrate for the first time that CH increases I(CI.Ca) density, which is attributable to an upregulation of TMEM16A expression in PASMCs. The augmented CaCC activity in PASMCs may potentiate membrane depolarization and L-type channel activation in response to vasoconstrictors and enhance pulmonary vasoreactivity in CHPH.

Alteration of pulmonary artery integrin levels in chronic hypoxia and monocrotaline-induced pulmonary hypertension.

BACKGROUND: Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats. METHODS AND RESULTS: Immunoblot showed increased α(1)-, α(8)- and α(v)-integrins, and decreased α(5)-integrin levels in PAs of both models. ß(1)- and ß(3)-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α(1)- and α(5)-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α(1)-ligand collagen IV. Moreover, α(1)-integrin binding hexapeptide GRGDTP elicited an enhanced Ca(2+) response, whereas the response to α(5)-integrin binding peptide GRGDNP was reduced in CH-PASMCs. CONCLUSION: Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression.

Cytochrome P450 Epoxygenase-Dependent Activation of TRPV4 Channel Participates in Enhanced Serotonin-Induced Pulmonary Vasoconstriction in Chronic Hypoxic Pulmonary Hypertension.

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic trpv4-/- mice. These results suggest that TRPV4 contributes to the enhanced 5-HT-induced vasoconstriction in chronic hypoxic PAs, in part via the CYP-EET-TRPV4 pathway. Our results further support the notion that manipulation of TRPV4 function may offer a novel therapeutic strategy for the treatment of hypoxia-related pulmonary hypertension.

Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.

AIMS: Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). METHODS AND RESULTS: PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. CONCLUSIONS: RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.

Classical transient receptor potential 1 and 6 contribute to hypoxic pulmonary hypertension through differential regulation of pulmonary vascular functions.

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity, and vascular remodeling, which are associated with alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries (PAs) of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout (Trpc1(-/-) and Trpc6(-/-)) mice compared with wild-type after exposure to 10% O(2) for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1(-/-) and Trpc6(-/-) mice. Small PAs of normoxic wild-type mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1(-/-) PAs, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6(-/-) PAs, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the augmented vasoreactivity was attenuated in Trpc1(-/-) and eliminated in Trpc6(-/-) PAs. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of microvessels were further suppressed in TRPC1-TRPC6 double-knockout mice. Our results, therefore, provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes, and that the presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension in the mouse model.

Clinical management of acute interstitial pneumonia: a case report.

We describe a 51-year-old woman who was admitted to hospital because of cough and expectoration accompanied with general fatigue and progressive dyspnea. Chest HRCT scan showed areas of ground glass attenuation, consolidation, and traction bronchiectasis in bilateral bases of lungs. BAL fluid test and transbronchial lung biopsy failed to offer insightful evidence for diagnosis. She was clinically diagnosed with acute interstitial pneumonia (AIP). Treatment with mechanical ventilation and intravenous application of methylprednisolone (80 mg/day) showed poor clinical response and thus was followed by steroid pulse therapy (500 mg/day, 3 days). However, she died of respiratory dysfunction eventually. Autopsy showed diffuse alveolar damage associated with hyaline membrane formation, pulmonary interstitial, immature collagen edema, and focal type II pneumocyte hyperplasia.