. Preservation of serotonin-mediated contractility in adult sheep pulmonary arteries following long-term high-altitude hypoxia. High Alt. Med. Biol. 12:253-264.-Long-term hypoxia (LTH) can increase serotonin (5-HT) signaling as well as extracellular calcium entry in adult rodent pulmonary arteries (PA), and 5-HT is associated with pulmonary hypertension. Because LTH, 5-HT, and calcium entry are related, we tested the hypothesis that LTH increases 5-HT-mediated PA contractility and associated calcium influx through L-type Ca 2 + channels, nonselective cation channels (NSCC), and reverse-mode sodium-Ca 2 + exchange. We performed wire myography and confocal calcium imaging on pulmonary arteries from adult ewes that lived near sea level or were maintained at high-altitude (3801 m) for *110 days. LTH did not increase the arterial medial wall thickness, nor did it affect the potency or efficacy for 5-HT-induced PA contraction. Ketanserin (100 nM), a 5-HT 2A antagonist, shifted the 5-HT potency to a far greater extent than 1 lM GR-55562, a 5-HT 1B/D inhibitor. These influences were unaffected by LTH. The rank order for reducing 5-

Antenatal maternal long-term hypoxia (LTH) can alter serotonin (5-HT) and calcium (Ca 2þ ) signaling in fetal pulmonary arteries (PAs) and is associated with persistent pulmonary hypertension of the newborn (PPHN). In humans, the antenatal maternal hypoxia can be secondary to smoking, anemia, and chronic obstructive pulmonary disorders. However, the mechanisms of antenatal maternal hypoxia-related PPHN are unresolved. Because both LTH and 5-HT are associated with PPHN, we tested the hypothesis that antenatal maternal LTH can increase 5-HT-mediated PA contraction and associated extracellular Ca 2þ influx through L-type Ca 2þ channels (Ca L ), nonselective cation channels (NSCCs), and reverse-mode sodium-calcium exchanger (NCX) in the near-term fetus. We performed wire myography and confocal-Ca 2þ imaging approaches on fetal lamb PA (*140 days of gestation) from normoxic ewes or those acclimatized to high-altitude LTH (3801 m) for *110 days. Long-term hypoxia reduced the potency but not the efficacy of 5-HT-induced PA contraction. Ketanserin (100 nmol/L), a 5-HT 2A antagonist, shifted 5-HT potency irrespective of LTH, while GR-55562 (1 mmol/L), a 5-HT 1B/D inhibitor, antagonized 5-HT-induced contraction in normoxic fetuses only. Various inhibitors for Ca L , NSCC, and reverse-mode NCX were used in contraction studies. Contraction was reliant on extracellular Ca 2þ regardless of maternal hypoxia, NSCC was more important to contraction than Ca L , and reverse-mode NCX had little or no role in contraction. Long-term hypoxia also attenuated the effects of 2-APB and flufenamic acid and reduced Ca 2þ responses observed by imaging studies. Overall, LTH reduced 5HT 1B/D function and increased NSCC-related Ca 2þ -dependent contraction in ovine fetuses, which may compromise pulmonary vascular function in the newborn.

Ryanodine is a selective ryanodine receptor (RyR) blocker, with binding dependent on RyR opening. In whole-cell studies, ryanodine binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum, which restricts studies of inositol-1,4,5-trisphosphate receptor (InsP 3 R) activity. Other RyR blockers have nonselective effects that also limit their utility. 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) blocks RyR-elicited Ca 2ϩ increases in skeletal and cardiac muscle; yet, its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP 3 Rs; thus, we tested the ability of FLA 365 to block RyR-and serotonin-mediated InsP 3 R-elicited Ca 2ϩ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca 2ϩ increases that were reversibly reduced by FLA 365, with an estimated IC 50 of ϳ1 to 1.5 M, and inhibited by 10 M ryanodine or 10 M cyclopiazonic acid. FLA 365 also blocked L-type Ca 2ϩ channel activity, with 10 M reducing Ba 2ϩ current amplitude in patch voltage-clamp studies to 54 Ϯ 6% of control and 100 M FLA 365 reducing membrane current to 21 Ϯ 6%. InsP 3 R-mediated Ca 2ϩ responses elicited by 10 M 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 M carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 M FLA 365, but they were reduced by 20 M FLA 365 to 76 Ϯ 9% of control in canine PASMCs and 52 Ϯ 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca 2ϩ channels or InsP 3 R in canine PASMCs.

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PKdeficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothe- The plasma kallikrein-kinin system (KKS) 2 consists of three proenzymes; factor XII (FXII, Hageman factor), prekallikrein (PK, Fletcher factor), and factor XI (FXI, plasma thromboplastin antecedent) as well as one cofactor; high molecular weight kininogen (HK, Fitzgerald factor). KKS is involved in the regulation of hemodynamics, inflammation, complement activation, angiogenesis, thrombosis, and fibrinolysis. Basically, all these proposed roles represent a range of overlapping effects that contribute to various extents toward vasodilation and healing. Therefore, the plasma KKS can be considered to have a spectrum of physiological effects, ranging at one extreme from a hemostatic state of vasodilation and promotion of smooth blood flow, all the way to a prothrombotic state. It is conceivable to suggest that other mechanisms proposed about respiratory, retinal, and renal systems can fit into this spectrum of physiological effects (1-3). There is accumulating evidence suggesting that when the plasma KKS is activated, the results are a sequential release of proteolytic enzymes and vasoactive peptides, generation of both angiogenic and anti-angiogenic molecules, stabilization of thrombus, and an increase in protease inhibitor activity in blood (4 -7). The activation of HK-PK complex on endothelial cells triggers vasodilation through smooth muscle relaxation, inhibits platelet aggregation, and induces proinflammatory responses. Of note, the direct assembly of HK, PK, and FXII on vascular smooth muscle cells (VSMC) also results in the activation of PK to kallikrein (8). The induction of these physiological reactions is caused by the release of the vasoactive peptide bradykinin (BK) from HK by kallikrein. Bradykinin B 2 receptor activation by BK mediates the activation of endothelial nitric-oxide synthase (eNOS) and phospholipase A 2 (PLA 2 ) leading to production of nitric oxide (NO) and prostacylin (PGI 2 ). Evidence suggests that BK phosphorylates p44/42 mitogen-activated protein kinase in VMSC, which is blocked by BK antagonist HOE-140 (8).Besides having a direct effect on blood vessels, the HK-PK complex has also been shown to mediate the effects of other pro-inflammatory molecules. Recent study suggests that the inhibitors of both BK and factor XII activity protect from mast cell-induced effects not only in patients but also in genetically engineered mouse models. The authors proposed that this class of inhibitors could be useful to treat allergic diseases (9)....

Chronic hypoxia (CH) can cause structural changes in the lung where the smooth muscle layer thickens due to myocyte hypertrophy and hyperplasia. CH compromises pulmonary arterial (PA) contractility and Ca2+‐signaling in the sheep fetus as well as the non‐pregnant ewe. Yet, the influence of CH on sheep PA and myocyte structure is unresolved, which is important as this relates to pulmonary pathologies. We therefore tested the hypothesis that CH thickens the smooth muscle layer and causes myocyte hypertrophy by evaluating the structure of PAs isolated from late‐gestational fetuses or adults maintained under normoxic conditions or exposed to CH by housing animals at 12,470 ft for ~ 110 days. To visualize smooth muscle cells, arterial segments were stained with anti‐α‐smooth muscle actin while nuclei were stained with DAPI. 3‐D images were made on a laser scanning confocal microscope by optical sample sectioning. The thickness of the smooth muscle layer was determined, as was myocyte and nuclear length, width, circumference, area and cell density. Fetal cells were smaller than adult and CH induced cellular hypertrophy in fetal and adult myocytes, although CH did not thicken the smooth muscle layer or alter cell density. Similarly, CH caused nuclear enlargement. Overall, these CH‐dependent changes in cell and nuclear morphometry are consistent with PA pathogenesis. Support from NSF, NIH, UM, LLUMC.