Phase Of Hypoxic Pulmonary Vasoconstriction Research Articles

Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release

Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 μm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 μm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10 μm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 μm) and attenuated by the protein kinase C inhibitor Gö6983 (3 μm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 μm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage-gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.

Gap junctions support the sustained phase of hypoxic pulmonary vasoconstriction by facilitating calcium sensitization.

AimsTo determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV).Methods and resultsStudies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F2α (PGF2α, 3 µM) or 20 mM K+. The GJ inhibitors 18β-glycyrrhetinic acid (18β-GA, 30 µM), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 µM) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca2+ sensitization. The voltage-dependent Ca2+ channel blocker diltiazem (10 µM) had no effect on HPV, and did not alter the inhibitory action of 18β-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca2+ sensitization, in the presence of high glucose 18β-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca2+ using Fura PE-3 demonstrated that whilst 18β-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca2+. Consistent with this, 18β-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18β-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response.ConclusionThese results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca2+ sensitization during sustained HPV, but not elevation of intracellular Ca2+, and may explain the dependence of the former on an intact endothelium.

Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction

The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30μM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5mg/kg) 30min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine, known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously (70mg/kg). We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release.

O2 sensing and Ca2+ release in hypoxic pulmonary vasoconstriction in non‐preconstricted pulmonary arteries from rats

We assessed the roles of Ca2+ release, the electron transport chain and NOX in hypoxic pulmonary vasoconstriction (HPV), the process by which hypoxia constricts pulmonary arteries (PA). HPV in the absence of pretone in 2nd and 3rd order rat PA branches gassed with 95% N2, 5% CO2 for 40 min demonstrated an initial transient tension increase (phase 1) followed by a slow rise in tension (phase 2). Phase 1 was abolished but phase 2 was unaffected by the complex I inhibitor rotenone (1 μM). Both phases of HPV were abolished by the complex IV inhibitor cyanide (10 μM). Pretreatment of PA with superoxide dismutase (200 units/ml), catalase (200 units/ml), their combination, or with the putative NOX inhibitor VAS 2870 (10 μM) had no effect on either phase of HPV. The superoxide scavenger TEMPOL (3mM) reduced phase 1 by 42% and abolished phase 2. 10 μM NED‐19 had no effect on either phase of HPV. The PKC blocker Gö6983 (3 μM) reduced both phases by ~30%. Pretreatment with 100 μM ryanodine reduced both phases 1 by ~70%. Pretreatment with a combination of ryanodine (10 μM) and caffeine (10 mM) to deplete SR Ca2+ abolished both phases. Applying zero Ca2+ ‐PSS ablated phase 1 HPV but had no effect on phase 2, and re‐adding Ca2+ during phase 2 did not increase contraction. These data indicate that O2 sensing mechanisms for phase 1 and 2 differ, but in neither case involve NOX. Phase 2 HPV is apparently fully accounted for by the release of Ca2+ from the SR.

Effect of Altitude on the Heart and the Lungs

This review focuses on the effects of altitude exposure from 1 to several days or weeks as occurs in tourists, trekkers, and mountaineers who visit high altitude and normally reside near sea level. We briefly review the acute physiological adjustments and early acclimatization that occur in the cardiovascular system and the lungs of healthy individuals. These ensure life-sustaining oxygen delivery to the tissues despite a reduction in the partial pressure of inspired oxygen between 20% and 60% at 2500 and 8000 m, respectively. One of the acute adjustments, hypoxic pulmonary vasoconstriction (HPV), may be disadvantageous in those with a vigorous response and lead to 2 potentially lethal illnesses, high-altitude pulmonary edema (HAPE) and subacute mountain sickness (SAMS), which we present in more detail. Finally, on the basis of knowledge about the acute physiological adjustments and acclimatization and, when available, a review of the literature, we discuss the high-altitude tolerance of patients with coronary artery disease, congestive heart failure, arrhythmias, systemic hypertension, and pulmonary hypertension. ### Circulation The major effects of acute hypoxia on the heart and lung are shown in Figure 1. Hypoxia directly affects the vascular tone of the pulmonary and systemic resistance vessels and increases ventilation and sympathetic activity via stimulation of the peripheral chemoreceptors.1 Interactions occur between the direct effects of hypoxia on blood vessels and the chemoreceptor-mediated responses in the systemic and pulmonary circulation. Figure 1. Effects of hypoxia on systemic and pulmonary circulation. Unraveling the underlying mechanisms of the hypoxic vasodilatation of systemic arterioles is an active area of research. Several mechanisms appear to regulate local oxygen delivery according to the needs of the tissues2,3; for instance, the release of ATP from red blood cells and the generation of NO by various ways appear to regulate local oxygen delivery according to the needs …

Role of glucose in pulmonary artery vasoconstriction during hypoxia

We have previously shown that the sustained phase of hypoxic pulmonary vasoconstriction (HPV) is critically dependent on extracellular glucose, though this does not seem to be related to the energy requirements of constriction per se (Leach et al, 2001, J Physiol 536, 211-224). As sustained HPV involves Ca2+ sensitisation, and changes in glucose preferentially affected force development rather than intracellular [Ca2+], we hypothesised that glucose was supporting Rho kinase dependent Ca2+ sensitisation. We examined the effects of varying glucose in small intrapulmonary arteries of the rat on MYPT-1 phosphorylation during HPV, and on the response to PGF2α (which also causes Ca2+ sensitisation) during hypoxia. After 45 min of hypoxia, MYPT-1 phosphorylation was significantly reduced in both 2.5mM (69 ± 11% of phosphorylation in 5mM, n=8, p<0.01) and 0mM glucose (19 ± 4%, n=8, p<0.01). During hypoxia, the concentration-response relationship for PGF2α was progressively suppressed as glucose was reduced (e.g. at 30μM PGF2α :- 5mM: 38 ± 4% of response to 80mM [K+]; 2.5mM: 28 ± 5%; 0mM: 9 ± 1%; n=6, p<0.05 for both). In the absence of extracellular Ca2+ reducing glucose also suppressed PGF2α :-induced tension (e.g. at 30μM PGF2α :- 5mM: 14 ± 1%; 2.5mM: 12 ± 1%; 0mM: 9 ± 1%; n=8, p<0.05 ANOVA). These data suggest that glycolysis is important for supporting Ca2+ sensitisation. Supported by the Wellcome Trust.

Role of src‐family kinases in hypoxic pulmonary vasoconstriction

We investigated the effects of inhibition of src-family kinases (srcFK) on both contraction and tyrosine phosphorylation in response to hypoxia in intrapulmonary artery (IPA) of rat. Isometric force was measured in isolated IPA mounted on a myograph in bicarbonate-buffered physiological salt solution (PSS, gassed with 5% CO2/95% air, at 37°C). In the presence of a low concentration of PGF2α, hypoxia (5% CO2/95% N2) induced a biphasic hypoxic pulmonary vasoconstriction (HPV). Both phases of HPV were inhibited by the srcFK blocker PP2 (30 μM; 1st phase, 40 ± 9 % inhibition, P<0.01; 2nd phase, 46 ±8 % inhibition, P<0.01, n = 11), whereas its inactive analogue, PP3 (30 μM) had no effect on HPV (n = 8). Tyrosine phosphorylation was measured (using SDS-PAGE Western Blot, mouse anti-phosphotyrosine primary, horse-radish peroxidase conjugated anti-mouse IgG secondary) in protein samples prepared from IPA treated with either hypoxia alone, or treated with hypoxia plus 30 μM PP2 and compared to untreated controls (in PSS, at 37°C). Several protein bands were observed, and in some of these, tyrosine phosphorylation was significantly increased following hypoxia, compared to controls (110–120 kD, 94 ± 19 % increase, P<0.05; 75 kD, 106 ± 30 % increase, P<0.05; 65–70 kD, 164 ± 65 % increase, P<0.05, 60 kD, 63 ± 22 % increase, P<0.05; n = 5–8). All increases were abolished by PP2. PGF2α (20μM) also increased tyrosine phosphorylation of the bands at 65–70 and 110–120 kD. Our results suggest that srcFK may be activated by hypoxia in IPA and that they may act upon multiple putative cellular targets to contribute to the contractile response Supported by Wellcome Trust 062554.

Two temporal components within the human pulmonary vascular response to ∼2 h of isocapnic hypoxia

The time course of the pulmonary vascular response to hypoxia in humans has not been fully defined. In this investigation, study A was designed to assess the form of the increase in pulmonary vascular tone at the onset of hypoxia and to determine whether a steady plateau ensues over the following approximately 20 min. Twelve volunteers were exposed twice to 5 min of isocapnic euoxia (end-tidal Po(2) = 100 Torr), 25 min of isocapnic hypoxia (end-tidal Po(2) = 50 Torr), and finally 5 min of isocapnic euoxia. Study B was designed to look for the onset of a slower pulmonary vascular response, and, if possible, to determine a latency for this process. Seven volunteers were exposed to 5 min of isocapnic euoxia, 105 min of isocapnic hypoxia, and finally 10 min of isocapnic euoxia. For both studies, control protocols consisting of isocapnic euoxia were undertaken. Doppler echocardiography was used to measure cardiac output and the maximum tricuspid pressure gradient during systole, and estimates of pulmonary vascular resistance were calculated. For study A, the initial response was well described by a monoexponential process with a time constant of 2.4 +/- 0.7 min (mean +/- SE). After this, there was a plateau phase lasting at least 20 min. In study B, a second slower phase was identified, with vascular tone beginning to rise again after a latency of 43 +/- 5 min. These findings demonstrate the presence of two distinct phases of hypoxic pulmonary vasoconstriction, which may result from two distinct underlying processes.

Hypoxia activates a background conductance in freshly isolated pulmonary arterial endothelial cells of the rat

Utilising the patch-clamp recording technique we have demonstrated for the first time the effects of hypoxia on the background current in pulmonary arterial endothelial cells. Electrophysiological studies revealed the presence of a novel oxygen-sensitive, non-selective cation conductance ( I NSC) in these cells. The inward component of I NSC was significantly potentiated by hypoxia. Both the inward and outward components of I NSC were inhibited by both La 3+ and Gd 3+. Hypoxic activation of I NSC may provide an important Ca 2+ influx pathway essential for the release of a pulmonary-selective vasoconstrictor pivotal to the sustained phase of hypoxic pulmonary vasoconstriction.

Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor.

1. The mechanisms responsible for sensing hypoxia and initiating hypoxic pulmonary vasoconstriction (HPV) are unclear. We therefore examined the roles of the mitochondrial electron transport chain (ETC) and glycolysis in HPV of rat small intrapulmonary arteries (IPAs). 2. HPV demonstrated a transient constriction (phase 1) superimposed on a sustained constriction (phase 2). Inhibition of complex I of the ETC with rotenone (100 nM) or complex III with myxothiazol (100 nM) did not cause vasoconstriction in normoxia, but abolished both phases of HPV. Rotenone inhibited the hypoxia-induced rise in intracellular Ca(2+) ([Ca(2+)](i)). Succinate (5 mM), a substrate for complex II, reversed the effects of rotenone but not myxothiazol on HPV, but did not affect the rise in NAD(P)H fluorescence induced by hypoxia or rotenone. Inhibition of cytochrome oxidase with cyanide (100 microM) potentiated phase 2 constriction. 3. Phase 2 of HPV, but not phase 1, was highly correlated with glucose concentration, being potentiated by 15 mM but abolished in its absence, or following inhibition of glycolysis by iodoacetate or 2-deoxyglucose. Glucose concentration did not affect the rise in [Ca(2+)](i) during HPV. 4. Depolarisation-induced constriction was unaffected by hypoxia except in the absence of glucose, when it was depressed by approximately 50 %. Depolarisation-induced constriction was depressed by rotenone during hypoxia by 23 +/- 4 %; cyanide was without effect. 5. Hypoxia increased 2-deoxy-[(3)H]glucose uptake in endothelium-denuded IPAs by 235 +/- 32 %, and in mesenteric arteries by 218 +/- 38 %. 6. We conclude that complex III of the mitochondrial ETC acts as the hypoxic sensor in HPV, and initiates the rise in smooth muscle [Ca(2+)](i) by a mechanism unrelated to changes in cytosolic redox state per se, but more probably by increased production of superoxide. Additionally, glucose and glycolysis are essential for development of the sustained phase 2 of HPV, and support an endothelium-dependent Ca(2+)-sensitisation pathway rather than the rise in [Ca(2+)](i).

Inhibition of hypoxic pulmonary vasoconstriction in isolated rat resistance arteries by atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) is a vasodilator secreted by the heart in response to right atrial stretch. We have hypothesized that ANP may be released to attenuate pulmonary hypertension due to hypoxia. We have examined whether ANP inhibits hypoxic pulmonary vasoconstriction (HPV) in isolated pulmonary resistance vessels (PRV) from chronically hypoxic (CH) rats, compared to air-breathing, control (C) rats. After at least 17 days of chronic hypoxia, vessels (n = 29) were dissected from CH and C littermates and mounted in an automated myograph. The inhibitory effect of ANP on the rapid first contractile phase of HPV, and the relaxant effect of ANP on vessels tonically contracted in the second phase of HPV, were studied. ANP caused concentration-dependent inhibition of HPV in both C and CH vessels (p < 0.001), whilst vehicle had no effect (mean maximum inhibition was 88 and 101%, respectively, at 17 nM ANP). ANP also caused significant concentration-dependent relaxation of the second contractile phase of HPV, which was similar in C and CH vessels (mean maximum relaxation of 89 and 94%, respectively; median effective concentrations were 2.4 and 2.0 nM, respectively). We conclude that atrial natriuretic peptide is a potent antagonist of both contractile phases of hypoxic pulmonary vasoconstriction in isolated rat pulmonary resistance vessels at concentrations similar to those observed in hypoxic pulmonary hypertension in life. There was no difference between vessels from chronically hypoxic and control animals.

Effect of the Calcium Antagonist Amlodipine on the Two Phases of Hypoxic Pulmonary Vasoconstriction in Rat Large and Small Isolated Pulmonary Arteries

Calcium antagonists inhibit hypoxic pulmonary vasoconstriction (HPV), which was recently shown to be biphasic in isolated vessels, but the sensitivity of the two phases to calcium antagonism is unknown. We studied HPV in rat isolated large pulmonary arteries (PA) and resistance pulmonary arteries (PRV), using a small vessel myograph. Two phases of acute hypoxic pulmonary vasoconstriction were confirmed to occur in both sizes of vessel. The first phase was short-lived and was followed by a more persistent phase of gradual onset. The magnitude of the first phase of contraction was greater than the second (p < 0.01). The first phase was reproducible, but the second was variable. Tension generated per square millimeter of vessel during both phases of hypoxic contraction was significantly greater in PRV than in PA (p < 0.05). Amlodipine caused dose-dependent inhibition of the first phase of HPV (p = 0.01), with a greater effect in PRV (p < 0.01). The second phase was not significantly inhibited. These results confirm that HPV is biphasic in isolated large and small PA, with a greater sensitivity of the first phase to calcium antagonism. Therefore, the first phase may represent HPV as observed in vivo.

Intense slow hypoxic pulmonary vasoconstriction in gas-filled and liquid-filled lungs: an in vivo study in the rabbit.

To examine the hypothesis that hypoxic pulmonary vasoconstriction may have a slower time course and greater intensity than is currently recognized, experiments were conducted in twelve anaesthetized rabbits subjected to unilateral lung hypoxia for 6 h. Endobronchial cannulation was used to maintain apnoea of one lung at constant airway pressure whilst inflating the lung with nitrogen or liquid. The second lung was ventilated with oxygen to maintain normocapnia and oxygen transfer. A pulmonary ventilated with oxygen to maintain normocapnia and oxygen transfer. A pulmonary artery catheter was introduced non-invasively. Pulmonary shunt was derived from mixed venous and arterial blood gas parameters. Pulmonary artery pressure was monitored continuously and cardiac output was estimated from oxygen uptake measurements before and after 6 h unilateral hypoxia. The experiments show that a rapid phase of hypoxic pulmonary vasoconstriction is followed by a slow phase which develops over hours. The slow phase is associated with a massive blood flow diversion from the hypoxic lung, such that pulmonary shunt after 6 h unilateral hypoxia is indistinguishable from baseline shunt during bilateral ventilation with oxygen. The response is reversible, but with a similarly slow time course. Results from nitrogen and liquid filling of the lung are similar. These findings are consistent with early experiments by Dirken and Heemstra in 1948 (Quart F Exp Physiol 34, 193-211), and challenge the prevailing notion that hypoxic pulmonary vasoconstriction is always a rapid and relatively weak physiological response to hypoxia.