Bitter taste receptors (TAS2Rs) of the tongue likely evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants, leading to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased ASM [Ca2+]i in a Gβγ, PLCβ and IP3-receptor dependent manner which would be expected (like acetylcholine) to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM, and dilation of airways that was 3-fold greater than β-agonists. Relaxation by TAS2Rs is from a localized [Ca2+]i response at the cell membrane which opens BKCa channels leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in an asthma mouse model. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

Chronic hypoxia induces polycythemia, pulmonary hypertension, right ventricular hypertrophy, and weight loss. Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that mediate adaptive responses to hypoxia, including erythropoietin, vascular endothelial growth factor, and glycolytic enzymes. Expression of the HIF-1α subunit increases exponentially as O 2 concentration is decreased. Hif1a -/-mouse embryos with complete deficiency of HIF-1α due to homozygosity for a null allele at the Hif1a locus die at midgestation, with multiple cardiovascular malformations and mesenchymal cell death. Hif1a +/-heterozygotes develop normally and are indistinguishable from Hif1a +/+ wild-type littermates when maintained under normoxic conditions. In this study, the physiological responses of Hif1a +/-and Hif1a +/+ mice exposed to 10% O 2 for one to six weeks were analyzed. Hif1a +/-mice demonstrated significantly delayed development of polycythemia, right ventricular hypertrophy, pulmonary hypertension, and pulmonary vascular remodeling and significantly greater weight loss compared with wild-type littermates. These results indicate that partial HIF-1α deficiency has significant effects on multiple systemic responses to chronic hypoxia.

Abstract-Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca 2ϩ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca 2ϩ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store-and receptor-operated Ca 2ϩ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca 2ϩ stores, caused dramatic increase in cation entry measured by Mn 2ϩ quenching of fura-2 and by Ca 2ϩ transients. OAG-induced responses were Ϸ700-fold more resistant to La 3ϩ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin-and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2-to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca 2ϩ or inhibition of store-operated Ca 2ϩ entry with La 3ϩ and SK&F-96365 reversed the elevated basal [Ca 2ϩ ] i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store-and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension. Key Words: pulmonary hypertension Ⅲ transient receptor potential channels Ⅲ store-operated Ca 2ϩ channels Ⅲ receptor-operated Ca 2ϩ channels P rolonged exposure to alveolar hypoxia causes pulmonary hypertension with profound vascular remodeling and increase in vasomotor tone. The increase in vascular tone is in part attributable to alterations in vasoconstricting and vasorelaxing influences imposed by the endothelially derived and circulating factors. 1 Recent evidence indicates that chronic hypoxia also causes intrinsic changes in ionic balance and Ca 2ϩ homeostasis in pulmonary arterial smooth muscle cells (PASMCs), including membrane depolarization, elevation in resting [Ca 2ϩ ] i , and changes in electrophysiological and Ca 2ϩ responses to vasoconstrictors and vasodilators. [2][3][4][5][6] The mechanism for alteration in Ca 2ϩ homeostasis in hypoxic PASMCs is controversial. Previous studies found significant suppression of voltage-gated K ϩ (K V ) currents and K V channel expression in PASMCs isolated fr...

Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.

In cross signaling between the ryanodine and DBP receptors in rat ventricular myocytes, independent of bulk cytosolic Ca2+ concentrations.MATERIALS AND METHODS Cell Isolation. Ventricular myocytes were enzymatically isolated from male Wistar rats (150-250 g) as described (15). In brief, rats were anesthetized (sodium pentobarbital, 30 mg/kg), and the excised hearts were perfused retrogradely through the aorta, first for 5 min with Ca2+-free Tyrode's solution and then with Ca2+-free Tyrode's solution containing collagenase (1.4 mg/ml; type A; Boehringer Mannheim) and protease (0.28 mg/ml; type XIV; Sigma), Myocytes were then dispersed in 0.2 mM Ca2+ Tyrode's solution and stored at room temperature for 1 hr before use.Measurements of Membrane Currents and Intracellular Ca2+ Transients. Myocytes were voltage clamped in the whole-cell clamp configuration. Membrane currents were recorded with a Dagan Instruments (Minneapolis) amplifier. Intracellular Ca2+ transients were simultaneously monitored with Ca2+-sensitive fluorescent dye (0.2 mM fura-2 or 0.1 mM indo-1) introduced into the myocytes through the patch pipettes (6, 16). The myocyte under examination was monitored continuously with a CCD television camera (Dage-MTI, Michigan City, IN) using red light. Ca2+ concentrations were calculated with the equation of Grynkiewicz et at (17) by assuming that the Kd values of fura-2 and indo-1 were 224 and 213 nM, respectively (17, 18). Indo-1 fluorescence was calibrated as described (16).Solutions. The modified Tyrode's solution contained 142 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, and 0.02 mM tetrodotoxin (TTX) at pH 7.4. The standard internal solution contained 110 mM CsCl, 10 mM NaCl, 5 mM MgATP, 10 mM Hepes, 20 mM Et4NCl, and 0.01 mM cAMP at pH 7.2. In the experiments in which we studied possible inactivation ofICa by SR Ca2+ release, 0.2 mM cAMP was added to the internal solution to optimize ICa and Ca2+ uptake.

Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation‐contraction coupling was investigated using a novel fluorescence method. Under whole‐cell voltage‐clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mm, 150 nm free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low‐affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA‐5N (OG‐5N; 1.0 mm, Kd≈ 31 μm), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+. Indeed, the OG‐5N transients in EGTA‐containing cells consisted of a prominent spike followed by a small pedestal. The OG‐5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model‐derived SR Ca2+ release function reported previously. In SR Ca2+‐depleted cells, the OG‐5N transient also closely followed the waveform of L‐type Ca2+ current (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa‐elicited OG‐5N signal. The OG‐5N image signal was localized to discrete release sites at the Z‐line level of sarcomeres, indicating that the local OG‐5N spike arises from ‘Ca2+ spikes’ at transverse (T) tubule‐SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules). Both peak SR release flux and total amount of released Ca2+ exhibited a bell‐shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mm s−1) at 0 mV; in contrast, maximal total release occurred at −20 mV (71 versus 61 μm at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole‐cell Ca2+ transient and the Ca2+‐myofilament interaction.

Transient receptor potential melastatin-(TRPM) and vanilloid-related (TRPV) channels are nonselective cation channels pertinent to diverse physiological functions. Multiple TRPM and TRPV channel subtypes have been identified and cloned in different tissues. However, their information in vascular tissue is scant. In this study, we sought to identify TRPM and TRPV channel subtypes expressed in rat deendothelialized intralobar pulmonary arteries (PAs) and aorta. With RT-PCR, mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of TRPV family were detected in both PAs and aorta. Quantitative real-time RT-PCR showed that TRPM8 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Moreover, Western blot analysis verified expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in both types of vascular tissue. To examine the functional activities of these channels, we monitored intracellular Ca 2ϩ transients ([Ca 2ϩ ]i) in pulmonary arterial smooth muscle cells (PASMCs) and aortic smooth muscle cells (ASMCs). The TRPM8 agonist menthol (300 M) and the TRPV4 agonist 4␣-phorbol 12,13-didecanoate (1 M) evoked significant increases in [Ca 2ϩ ]i in PASMCs and ASMCs. These Ca 2ϩ responses were abolished in the absence of extracellular Ca 2ϩ or the presence of 300 M Ni 2ϩ but were unaffected by 1 M nifedipine, suggesting Ca 2ϩ influx via nonselective cation channels. Hence, for the first time, our results indicate that multiple functional TRPM and TRPV channels are coexpressed in rat intralobar PAs and aorta. These novel Ca 2ϩ entry pathways may play important roles in the regulation of pulmonary and systemic circulation. transient receptor potential channels; calcium signaling; nonselective cation channels THE TRANSIENT RECEPTOR POTENTIAL (TRP) channels belong to a large superfamily of cation channels that have diverse physiological functions in both nonexcitable and excitable cells (26,33). On the basis of sequence homology, the TRP superfamily can be divided into three major subfamilies of canonical (TRPC), melastatin-related (TRPM), and vanilloid-related (TRPV) channels, as well as four more distant subfamilies of polycystin-related (TRPP), mucolipin-related (TRPML), ankyrin-related (TRPA), and no mechanoreceptor potential C, or NOMPC (TRPN), channels and/or proteins.The classic or canonical TRPC subfamily consists of seven members (TRPC1-7), which have attracted enormous attention because of their putative roles as store-operated and receptoroperated cation channels. In vascular smooth muscle, it is generally accepted that TRPC1 channels are related to storeoperated Ca 2ϩ entry, which can be activated by depletion of Ca 2ϩ stores (1,4,17,22,41,48), whereas TRPC6 (and TRPC3) channels are involved in receptor-operated Ca 2ϩ entry, which can be activated directly by diacylglycerol in a PKC-independent manner (13,15,18,22). TRPC channels have been shown to play pivotal roles in vasoconstriction induced by ␣-adrenoceptor agonists, vasopressin, endo...

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).