James S K Sham

Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α

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-1alpha subunit increases exponentially as O2 concentration is decreased. Hif1a-/- mouse embryos with complete deficiency of HIF-1alpha 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% O2 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-1alpha deficiency has significant effects on multiple systemic responses to chronic hypoxia.

Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Bitter taste receptors (TAS2Rs) on the tongue probably 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 that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2+]i) in ASM in a Gβγ–, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP3) receptor–dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2+]i response at the cell membrane, which opens large-conductance Ca2+-activated K+ (BKCa) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. 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-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells : A novel mechanism of hypoxic pulmonary hypertension

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ 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 Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca2+]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.

Direct measurement of SR release flux by tracking ‘Ca2+ spikes’ in rat cardiac myocytes

1 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. 2 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+. 3 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. 4 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). 5 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.

Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1

Although endothelin (ET)-1 is an important regulator of pulmonary vascular tone, little is known about the mechanisms by which ET-1 causes contraction in this tissue. Using the whole cell patch-clamp technique in rat intrapulmonary arterial smooth muscle cells, we found that ET-1 and the voltage-dependent K+(KV)-channel antagonist 4-aminopyridine, but not the Ca2+-activated K+-channel antagonist charybdotoxin (ChTX), caused membrane depolarization. In the presence of 100 nM ChTX, ET-1 (10-10to 10-7 M) caused a concentration-dependent inhibition of K+ current (56.2 ± 3.8% at 10-7 M) and increased the rate of current inactivation. These effects of ET-1 on K+ current were markedly reduced by inhibitors of protein kinase C (staurosporine and GF 109203X) and phospholipase C (U-73122) or under Ca2+-free conditions and were mimicked by activators of protein kinase C (phorbol 12-myristate 13-actetate and 1,2-dioctanoyl- sn-glycerol). These data suggest that ET-1 modulated pulmonary vascular reactivity by depolarizing pulmonary arterial smooth muscle, due in part to the inhibition of KV current that occurred via activation of the phospholipase C-protein kinase C signal transduction pathway.Although endothelin (ET)-1 is an important regulator of pulmonary vascular tone, little is known about the mechanisms by which ET-1 causes contraction in this tissue. Using the whole cell patch-clamp technique in rat intrapulmonary arterial smooth muscle cells, we found that ET-1 and the voltage-dependent K+ (Kv)-channel antagonist 4-aminopyridine, but not the Ca(2+)-activated K(+)-channel antagonist charybdotoxin (ChTX), caused membrane depolarization. In the presence of 100 nM ChTX, ET-1 (10(-10) to 10(-7) M) caused a concentration-dependent inhibition of K+ current (56.2 +/- 3.8% at 10(-7) M) and increased the rate of current inactivation. These effects of ET-1 on K+ current were markedly reduced by inhibitors of protein kinase C (staurosporine and GF 109203X) and phospholipase C (U-73122) or under Ca(2+)-free conditions and were mimicked by activators of protein kinase C (phorbol 12-myristate 13-actetate and 1,2-dioctanoyl-sn-glycerol). These data suggest that ET-1 modulated pulmonary vascular reactivity by depolarizing pulmonary arterial smooth muscle, due in part to the inhibition of Kv current that occurred via activation of the phospholipase C-protein kinase C signal transduction pathway.

Neuropilin-1 Regulates Vascular Endothelial Growth Factor-Mediated Endothelial Permeability

Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.

Ca2+ release-induced inactivation of Ca2+ current in rat ventricular myocytes: evidence for local Ca2+ signalling.

1. Inactivation of Ca2+ current (ICa) induced by Ca2+ release from sarcoplasmic reticulum (SR) was studied in single rat ventricular myocytes using whole‐cell patch‐clamp and indo‐1 fluorescence measurement techniques. 2. Depolarizing pulses to 0 mV elicited large Ca2+ transients and ICa with biexponential inactivation kinetics. Varying SR Ca2+ loading by a 20 s pulse of caffeine showed that the fast component of ICa inactivation was dependent on the magnitude of Ca2+ release. 3. Inactivation of ICa induced by Ca2+ release was quantified, independently of voltage and Ca2+ entry, using a function termed fractional inhibition of ICa (FICa). The voltage relation of FICa had a negative slope, resembling that of single‐channel Ca2+ current (iCa) rather than the bell‐shaped current‐voltage (I‐V) relation of macroscopic ICa and Ca2+ transients. 4. Intracellular dialysis of myocytes with 10 mM EGTA (150 nM free [Ca2+]) had no effect on ICa inactivation induced by Ca2+ release, despite abolition of Ca2+ transients and cell contraction. Dialysis with 3 or 10 mM BAPTA (180 nM free [Ca2+]) attenuated FICa in a concentration‐dependent manner, with greater inhibition at positive than at negative potentials, consistent with more effective buffering of Ca2+ microdomains of smaller iCa. 5. Spatial profiles of [Ca2+] near an opened Ca2+ channel were simulated. [Ca2+] reached submillimolar levels at the mouth of the channel, and dropped steeply as radial distance increased. At any given distance from the channel, [Ca2+] was higher at negative than at positive potentials. The radii of Ca2+ microdomains were significantly reduced by 3 or 10 mM BAPTA, but not by 10 mM EGTA. 6. In conclusion, the distinctive voltage dependence and susceptibility of Ca2+ release‐induced ICa inactivation to fast and slow Ca2+ buffers suggests that the process is mediated through local changes of [Ca2+] in the vicinity of closely associated Ca2+ channels and ryanodine receptors.

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

•  A prolonged reduced oxygen level in the lungs, as occurs in patients of many chronic lung diseases and in residents living at high altitude, causes pulmonary hypertension characterized by profound structural and functional changes in pulmonary vasculature. •  Many of these changes are ascribed to alterations in Ca2+ homeostasis related to cation channels of pulmonary arterial smooth muscle cells. •  Here we report the increase of an anion conductance called calcium‐activated chloride channel and the expression of the channel gene TMEM16A in pulmonary arterial smooth muscle cells isolated from rats exposed to 10% oxygen for 3–4 weeks. •  The upregulation of the chloride channel contributes to the hyper‐responsiveness of pulmonary arteries to serotonin associated with pulmonary hypertension. •  These results help us to appreciate the importance of anion channels in the pathophysiology of pulmonary hypertension, and may lead to alternative strategies for the treatment of the disease.

Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes.

We tested the hypothesis that chronic hypoxia alters the regulation of K+ channels in intrapulmonary arterial smooth muscle cells (PASMCs). Charybdotoxin-insensitive, 4-aminopyridine-sensitive voltage-gated K+ (K(V,CI)) and Ca2+-activated K+ (KCa) currents were measured in freshly isolated PASMCs from rats exposed to 21 or 10% O2 for 17-21 days. In chronically hypoxic PASMCs, K(V, CI) current was reduced and KCa current was enhanced. 4-Aminopyridine (10 mM) depolarized both normoxic and chronically hypoxic PASMCs, whereas charybdotoxin (100 nM) had no effect in either group. The inhibitory effect of endothelin (ET)-1 (10(-7) M) on K(V,CI) current was significantly reduced in PASMCs from chronically hypoxic rats, whereas inhibition by angiotensin (ANG) II (10(-7) M) was enhanced. Neither ET-1 nor ANG II altered K(Ca) current in normoxic PASMCs; however, both stimulated K(Ca) current at positive potentials in chronically hypoxic PASMCs. These results suggest that although modulation of K(V,CI) and KCa channels by ET-1 and ANG II is altered by chronic hypoxia, the role of these channels in the regulation of resting membrane potential was not changed.