Antenatal chronic hypoxia can lead to pulmonary hypertension in newborns or more severe vascular remodeling and dangerous elevations in pressure. We have previously shown that antenatal chronic hypoxia deregulates Ca2+ spark activity, which are important to pulmonary vasodilation at birth. Chronic hypoxia can also increase oxidative stress, which can impair Ca2+ spark activity. In this research, we examined the impact of oxidative stress on Ca2+ spark activity, which other laboratories have shown to increase Ca2+ spark activity. We exposed pulmonary arteries from normoxic fetal sheep to reactive oxygen species (ROS) to mimic oxidative stress such as might occur with acute hypoxic stress by treating them with tert‐butyl H2O2 and reduced ROS with the antioxidant N‐Acetyl‐L‐Cysteine (NAC). Using customized analysis software, roughly 7 % of control cells had Ca2+ sparks and the frequency was 0.07 sparks/100 μm/sec. Spark activity was maintained in the presence of tert‐butyl H2O2 and NAC. The spatial and temporal aspects to the Ca2+ sparks were then determined. Spark amplitudes were unaffected as was the width. However, Ca2+ spark decay was increased slightly by tert‐butyl H2O2. Overall, oxidative stress has only a mild influence on Ca2+ spark activity on fetal pulmonary arteries from normoxic sheep.Grant Funding Source: Supported by NSF MRI 923559, NIH HD69746, P01HD31226, R01HD3807, LLUSOM, APS Frontiers in Physiology

Ryanodine receptor (RyR) activity causes Ca2+ sparks in cerebral arterial myocytes by activating adjacent Ca2+‐activated K+ (BK) channels, which can dilate arteries. Previous studies show ontogeny increases Ca2+ sparks and long‐term hypoxia (LTH) increases BK channel activity. We hypothesized that BK channel activity is greater in arterial myocytes of the LTH fetus than adult due to greater spark activity, and because BK channel clustering is greater in regions where sparks occur. To address this hypothesis we examined spark activity and BK channel clustering to sites of potential spark activity. This was accomplished using line‐scan and immunofluorescence techniques, respectively, in basilar arteries from LTH fetal (FH) and adult sheep (AH) kept at 3,801 m for > 100 days. The percentage of myocytes with sparks was ~ 2‐fold greater in FH versus AH and unaffected by 30 mM K (30K). Spark amplitude was 7% greater in FH 30K versus AH 30K, while no spatial or temporal changes to sparks were observed. BK channels were 2‐3 times more clustered and ~1.5 times more co‐localized with cholera toxin B‐labeled clusters in FH. These data suggest greater BK targeting to potential sites of spark activity in FH myocytes. Elevated RyR‐dependent Ca2+ spark activity in FH and altered patterns of BK distribution shed new light on the therapeutic potential of RyRs and BKs for treatment of cerebral vascular disease in newborns and adults.Grant Funding Source: Supported by NSF MRI 0923559, NIH HD‐069746, P01HD031226, R01HD003807, LLUSOM

High altitude is a risk factor in the development of pulmonary hypertension (PH). Treatment often includes the use of phosphodiesterase (PDE) inhibitors of PDE3 (milrinone, cAMP) and PDE5 (sildenafil, cGMP), which increase cyclic nucleotides and promotes vasodilation in pulmonary arterial (PA) myocytes. With respect to the current studies, c‐AMP and c‐GMP enhance ryanodine receptor (RyR) and Sarco/Endoplasmic Reticulum Ca2+ ‐ATPase (SERCA) pump activity in myocytes, which regulate intracellular Ca2+. To test whether long term hypoxia (LTH) impairs, and maturity enhances the effects of c‐AMP and c‐GMP on myocyte Ca2+ waves, we selected 3‐isobutyl‐1‐methyl xanthine (IBMX), a PDE inhibitor, as well as 8‐Br‐c‐AMP and 8‐Br‐c‐GMP. We measured cytosolic Ca2+ in PA myocytes of fetal and adult sheep that lived at low (~353 m) or high altitude (~3801 m) via confocal imaging of fluo‐4. Maturation increased while IBMX, 8‐Br‐c‐AMP, and 8‐Br‐c‐GMP each failed to alter the percentage of responsive cells independent of altitude. LTH fetuses had reduced wave amplitude and area under the curve (AUC), but wave kinetics were unaltered. The AUC was maintained in LTH adults, but waves were faster and larger. c‐GMP increased AUC by lengthening waves in all groups except for hypoxic fetuses. Alterations of Ca2+ regulatory pathways including SERCAs and RyRs could contribute to LTH impairment in Ca2+ waves and c‐GMP amplified signals.Grant Funding Source: Supported by NSF MRI 0923559, NIH HD‐069746, P01HD031226, , R01HD003807, 5P20 MD‐006988,LLUSOM

Ryanodine receptors (RyRs) are sensitive to reactive oxygen species in that this stress increases leak of Ca2+ through the channels. Our recent work shows RyR‐generated Ca2+ sparks are aberrant following prenatal chronic hypoxia (CH) in pulmonary arterial myocytes of fetal sheep. Other evidence suggests that the enhanced Ca2+ leak due to oxidative stress can cause loss of Ca2+ from the sarcoplasmic reticulum (SR); our Ca2+ spark data are consistent with such a loss in SR Ca2+ and CH can cause oxidative stress. To address potential Ca2+ loss we quantified 10 mM caffeine (CAF) elicited Ca2+ release from the SR, as RyR activation provides an index of Ca2+ storage. This was performed in pulmonary arterial myocytes of intact arteries using Fluo‐4 and confocal imaging approaches of fetal sheep that lived in normoxia at low altitude (FN) or fetal sheep that lived in CH at (3,801 m) for >100 days (FH). Using customized analysis software, the data show that under control conditions prenatal CH does not impact the area under the curve (AUC) due to CAF. Thus, the changes in spark activity previously observed likely arise from something other than alterations in SR Ca2+ storage. Pretreating PA with 1 mM tert‐butyl H2O2 resulted in a ~ 31% decrease in CAF‐elicited AUC in FN and a 6% increase in FH. These data suggest that sheep exposed to prenatal CH may adapt, which limits decrements in total SR Ca2+ storage in an attempt to preserve SR function.Grant Funding Source: NSF MRI 0923559, NIH HD‐069746, P01HD031226, R01HD003807, 5P20 MD‐006988, LLUSOM

Calcium sparks are due to ryanodine receptor (RyR) activation and are intimate to local and global calcium signals in pulmonary arterial (PA) myocytes. RyRs are heavily regulated and cyclic nucleotides, including cAMP and cGMP, can increase RyR activity and thereby augment Ca 2+ sparks and global Ca 2+ waves. One possibility is that enhanced RyR activity contributes to altered pulmonary arterial reactivity responses found in sheep born at high altitude. Yet, the influence of chronic hypoxia (CH) on cyclic nucleotide regulation of RyRs is unknown. In these studies, we tested the hypothesis that cAMP and cGMP increase Ca 2+ sparks and waves in PA myocytes from hypoxic sheep. Spark and wave activity was determined by visual analysis of line‐scan confocal recordings of Fluo‐4 or Fluo‐8 loaded PA from term‐fetal, ~ 10 day old, or adult sheep that lived at 3,200 meters for <100 days. Similar percentages of fetal, newborn, and adult myocytes had sparks and waves before cyclic nucleotide treatment, although newborns had more sparks per recording. cAMP and cGMP increased the number of adult cells with sparks and waves, yet, they respectively decreased the number of newborn and fetal myocytes with sparks. Spark activity was increased in adult myocytes by cGMP. The data suggest that cyclic nucleotides cause divergent regulation of RyRs from PA myocytes of immature CH sheep. NSF MRI 092355 (SMW), NIH P01HD031226, R01HD003807 (LDL)

Antenatal maternal long-term hypoxia (LTH) can alter serotonin (5-HT) and calcium (Ca2+) 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 Ca2+ influx through L-type Ca2+ 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-Ca2+ imaging approaches on fetal lamb PA (similar to 140 days of gestation) from normoxic ewes or those acclimatized to high-altitude LTH (3801 m) for similar to 110 days. Long-term hypoxia reduced the potency but not the efficacy of 5-HT-induced PA contraction. Ketanserin (100 nmol/L), a 5-HT2A antagonist, shifted 5-HT potency irrespective of LTH, while GR-55562 (1 mu mol/L), a 5-HT(1B/D)w 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 Ca2+ 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 Ca2+ responses observed by imaging studies. Overall, LTH reduced 5HT(1B/D) function and increased NSCC-related Ca2+-dependent contraction in ovine fetuses, which may compromise pulmonary vascular function in the newborn.

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 Ca2+ channels, nonselective cation channels (NSCC), and reverse-mode sodium-Ca2+ 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-HT2A antagonist, shifted the 5-HT potency to a far greater extent than 1 μM GR-55562, a 5-HT1B/D inhibitor. These influences were unaffected by LTH. The rank order for reducing 5-HT-induced PA contraction in normoxic animals was extracellular calcium removal≈10 mM Ni2+≈10 μM verapamil≈10 μM nifedipine with 50 μM SKF 96365>30 μM KB-R7943≈100 μM flufenamic acid≈10 μM nifedipine≈100 μM Gd3+> 100 μM La3+>500 μM Ni2+≈10 μM diltiazem≈50 μM 2-APB≈100 μM LOE 908. Contraction was not reduced by 100 μM spermine or 30 μM SN-6. LTH increased the effects of KB-R7943 and mitigated those of nifedipine but did not affect calcium responses in imaging studies. Overall, in adult sheep, arterial structure and 5-HT2A and 5HT1B/D functions are preserved following LTH while the role of NSCC-related calcium-dependent contraction is increased. These elements indicate preservation of PA contractility in LTH with minimal functional changes.

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 PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca2+](i) level in HPAEC. Gd3+ and verapamil potentiated the HK-induced increase in [Ca2+](i). HK-induced Ca2+ increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B-2 receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.

This study demonstrates that the bradykinin (BK) precursor, non‐enzymatic cofactor high molecular weight kininogen (HK), regulates endothelial function. HK circulates in complex with two different zymogens, namely, prekallikrein (PK) and factor XI (FXI). Both PK and FXI participate in the contact phase of blood coagulation through their binding to HK. The assembly and activation of the HK‐PK complex on endothelial cells results in the generation of BK and nitric oxide (NO). Since no clinical abnormality is apparent in patients with PK deficiency, investigations were performed to determine the functional role of HK on endothelial cells using a flow‐based assay. HK positively regulated endothelial cell function by modulating Ca2+‐dependent process and endothelial nitric oxide synthase (eNOS) function. The inhibitors of BK‐dependent signaling pathway impaired HK‐mediated signal transduction in endothelial cells. HKH20 and HOE140 significantly reduced HK‐ or HK/PK‐induced intracellular [Ca2+]i changes. This is a novel finding indicating that HK contributes to the maintenance of endothelial barrier function in the absence of PK. Taken together, these findings highlight a novel potential function for HK‐receptor‐mediated crosstalk in regulating endothelial cells and suggest that HK may serve as a cardioprotective peptide. This work was supported by NSF MRI 0619774, AHA and NCRR/NIH P20RR021929 to ZSM.

The pulmonary vasculature regulates lung blood flow in order to maintain blood oxygenation. However, chronic hypoxia (CH) such as that induced by high‐altitude exposure leads to changes and dysfunctions in the pulmonary vasculature. Serotonin (5‐HT) is an inflammatory mediator that triggers cytosolic Ca2+ increases and pulmonary arterial smooth muscle cell (PASMC) contractility. We have recently shown maturation and CH‐stress alters Ca2+ ‐dependent contractility in pulmonary arteries from sheep. The present study extends these findings by testing two hypotheses, these being that maturation enhances while CH reduces 5‐HT ‐generated Ca2+ signaling in sheep PASMCs. These hypotheses were assessed by performing confocal fluorescence microscopy of fluo‐4 in PASMCs in‐situ from fetal and adult sheep that were housed under normoxic conditions or at 12,470 feet (CH) for ~ 110 days. Basal Ca2+ activity was greater in adult than fetus and in CH‐adults as compared to their normoxic counterparts. In the presence of 10 μM 5‐HT, adult PASMCs from CH and normoxic sheep had similar firing rates. However, 5‐HT‐mediated cell firing was blunted in PASMCs from CH fetus. Maturation of PASMCs from normoxic sheep resulted in increased basal and 5‐HT‐elicited Ca2+ ‐ reactivity. These findings provide the first evidence that maturation and CH interact to alter 5‐HT‐dependent Ca2+ signaling. Support from NSF, NIH, UM, and LLUMC.

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.

Plasma kallikrein‐kinin system (KKS) is activated upon binding to subendothelium or endothelium, leading to the reduction of the concentrations of plasma prekallikrein (PK), factor XII, and high molecular weight kininogen (HK). Upon binding, PK converts to kallikrein on HK bound to endothelium. Kallikrein then liberates bradykinin from HK to activate bradykinin B2‐ and bradykinin B1 receptors that mediates nitric oxide and prostacyclin formation. However, the nature of KKS activation and signaling on endothelium remains divisive. We determined KKS activation and Ca2+ signaling in Fluo‐4 (10 μM) loaded cultured endothelial cells and by performing in‐situ recordings of endoethlial cells in mouse pulmonary arteries using confocal laser microscopy techniques. Stimulation of endothelial cells with HK or the complex of HK‐PK increased [Ca2+]i in a majority but not all cells, suggesting the involvement of a distinct physiologic mechanism(s). HK as well as HK‐PK concentration and changes in [Ca2+]i correlated and were inter‐dependent. HK‐induced changes in [Ca2+]i was significantly lower than the changes triggered by the complex of HK‐PK in endothelial cells. The [Ca2+]i was unaltered when HK binding to endothelium was blocked. These results indicate that a novel KKS cell‐signaling pathway regulates endothelial function. Support from NSF and AHA.

Serotonin (5HT) is important to pulmonary arterial (PA) reactivity and alterations in 5HT signaling are implicated in pulmonary hypertension in newborns as well as adults. Several reports also provide evidence for alterations in 5HT mediated contractility with maturation and in response to chronic hypoxia (CH). Further to this 5HT2A receptor activation and Ca2+ signaling is important to the contractility responses induced by 5HT, and we have evidence that Ca2+‐dependent contractility is reduced with CH in PA from sheep fetus. To delineate the cellular mechanisms associated with the loss in Ca2+‐dependent contractility we tested the hypothesis that 5HT generated Ca2+‐signaling is reduced with CH in fetal sheep. This was examined using in‐situ confocal imaging of Flou‐4 in PA isolated from CH fetus and adult. PA myocytes of CH fetal and adult had spontaneous cytosolic Ca2+ elevations and 10 μM 5HT induced cytosolic Ca2+ elevations in myocytes of fetal and adult sheep. The frequency of 5‐HT‐mediated Ca2+ oscillations in responsive cells was 0.01 ± 0.002 Hz in fetus and 0.021 ± 0.002 Hz in adult while the density of cells exhibiting Ca2+ elevations was 0.003 ± 0.001 cells/mm2/sec in fetus and 10‐fold greater in adult, being 0.03 ± 0.003 cells/mm2/sec. The reduced 5HT mediated Ca2+ signaling in fetal cells may underlie the loss of Ca2+‐dependent contractility previously observed. (Support from NSF, NIH and UM)

Muscarinic acetylcholine (ACh) receptor (mAChR) activation relaxes arteries through endothelium‐dependent NO signaling pathways and endothelium removal from most systemic arteries alleviates ACh mediated relaxation. However, Ach induces “paradoxical” contractility in endothelium disrupted pulmonary arteries (PA), through mAChR activation in multiple species including human. Chronic hypoxia (CH) disrupts vascular endothelium, producing loss of endothelium‐dependent PA relaxation. Given the intimacy between endothelial and myocyte function the hypothesis that CH reduces ACh‐dependent PA contractility was tested by performing wire‐myography of endothelium‐denuded PA rings from normoxic fetal and adult animals. The data show that Ach (100 μM) contracted arteries pre‐contracted with 125 mM KCl and this contractility was reduced by CH in adult PA and ablated in fetus. In CH adult, the mAChR agonist carbachol (CCh, 10 μM) caused contraction while 1 μM atropine (mAChR antagonist) blocked Ach and CCh contractility. Consistent with mAChR activation, In situ confocal microscopy approaches show Ach caused cytosolic Ca2+ increases in PA myocytes of CH adult. In conclusion, this study provides evidence for CH induced loss of mAChR PA contractility, a process that likely helps match ventilation to perfusion, especially as ACh causes bronchoconstriction. (Support from NIH, NSF and UM)

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 (InsP3R) 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 Ca2+ 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 InsP3Rs; thus, we tested the ability of FLA 365 to block RyR- and serotonin-mediated InsP3 R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of ∼1 to 1.5 μM, and inhibited by 10 μM ryanodine or 10 μM cyclopiazonic acid. FLA 365 also blocked L-type Ca2+ channel activity, with 10 μM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 ± 6% of control and 100 μM FLA 365 reducing membrane current to 21 ± 6%. InsP3R-mediated Ca2+ responses elicited by 10 μM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 μM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 μM FLA 365, but they were reduced by 20 μM FLA 365 to 76 ± 9% of control in canine PASMCs and 52 ± 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca2+ channels or InsP3R in canine PASMCs.