HPV (hypoxic pulmonary vasoconstriction) is the critical and distinguishing characteristic of the arteries that feed the lung. In marked contrast, systemic arteries dilate in response to hypoxia to meet the metabolic demands of the tissues they supply. Physiologically, HPV contributes to ventilation–perfusion matching in the lung by diverting blood flow to oxygen-rich areas. However, when alveolar hypoxia is global, as in diseases such as emphysema and cystic fibrosis, HPV leads to HPH (hypoxic pulmonary hypertension) and right heart failure. HPV is driven by the intrinsic response to hypoxia of two different cell types, namely the pulmonary arterial smooth muscle and endothelial cells. These are representatives of a group of specialized cells, commonly referred to as oxygen-sensing cells, which are defined by their acute sensitivity to relatively small changes in PO2 and have evolved to monitor oxygen supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport oxygen. Upon exposure to hypoxia, mitochondrial oxidative phosphorylation is inhibited in all such cells and this, in part, mediates cell activation. In the case of pulmonary arteries, constriction is triggered via: (i) calcium release from the smooth muscle sarcoplasmic reticulum and consequent store-depletion-activated calcium entry into the smooth muscle cells and, (ii) the modulation of transmitter release from the pulmonary artery endothelium, which leads to further constriction of the smooth muscle by increasing the sensitivity of the contractile apparatus to calcium.
Awareness of HPV (hypoxic pulmonary vasoconstriction) dawned in 1894 when Bradford and Dean  recorded a rise in pulmonary arterial pressure upon asphyxia. However, it was another 50 years before von Euler and Liljestrand  showed that hypoxia without hypercapnia induced constriction within the pulmonary circulation. In fact, von Euler and Liljestrand were the first to hypothesize that HPV may aid ventilation–perfusion matching, by diverting blood flow away from poorly ventilated areas of the lung. Thus HPV came to be recognized as the critical and distinguishing characteristic of pulmonary arteries. In contrast, systemic arteries dilate in response to tissue hypoxaemia, in order to match local perfusion to local metabolism.
HPV is not mediated by the autonomic nervous system
Investigations on the innervation of the pulmonary vasculature date back to the work of Bradford and Dean , which demonstrated that gross excitation of the spinal cord caused vasorelaxation within the systemic circulation, but had no effect on the pulmonary circulation. Significant progress on the basic mechanism of HPV was then provided by an investigation that suggested that HPV was a local response largely, or entirely, independent of the autonomic nervous system . The limited influence on HPV of the innervation within the pulmonary vasculature was confirmed by denervation. Thus HPV was permitted after chemical sympathectomy (using 6-hydroxydopamine), surgical denervation of the carotid and aortic chemoreceptors  and after bilateral cervical vagotomy . Most significantly, perhaps, bilateral lung transplants established that HPV remained unaffected following denervation in man . We can conclude, therefore, that neither central nor local regulation of the autonomic nervous system plays a role in mediating HPV.
Airway PO2 determines pulmonary vascular perfusion pressure at the level of pre-capillary resistance arteries
In 1951, Duke  demonstrated that HPV was not induced when the lung was perfused with hypoxic blood at a constant, normoxic alveolar oxygen tension. Later work  confirmed that the PO2 of the perfusate was not the determining factor, but instead that a fall in alveolar PO2 consistently triggered a pronounced increase in pulmonary vascular perfusion pressure. Clearly, therefore, HPV is triggered by a reduction in oxygen supply to the airways and/or the alveoli. This is consistent with the findings of Kato and Staub  who demonstrated, using unilobar hypoxia, that the small pre-capillary resistance arteries contributed most to the increase in pulmonary vascular perfusion pressure during alveolar hypoxia. Furthermore, it has been shown that the magnitude of HPV in isolated pulmonary arteries is inversely related to pulmonary artery diameter . It seems likely, therefore, that the principal mechanism(s) that drives HPV would offer a degree of selectivity for small compared with large pulmonary arteries, coupled with an even greater degree of selectivity for pulmonary versus systemic arteries.
Characteristics of the hypoxic response in isolated pulmonary arteries
In isolated pulmonary arteries, HPV is biphasic when induced by switching from a normoxic to a hypoxic gas mixture (Figure 1). Thus hypoxia induces an initial transient constriction (Phase 1) and a slow tonic constriction (Phase 2) [10,11]. Both phases of constriction are superimposed upon each other, i.e. they are initiated immediately on exposure to hypoxia. The initial transient constriction peaks within 5–10 min of the hypoxic challenge, while the underlying, tonic constriction peaks after 30–40 min [12,13]. In the isolated and perfused rat lung, however, a monophasic and sustained increase in perfusion pressure is induced by hypoxia, which has led to the physiological role of the transient, Phase 1 constriction of isolated pulmonary arteries being questioned [12,14].
HPV is mediated by mechanisms intrinsic to pulmonary arterial smooth muscle and endothelial cells
Consideration of the full facts available can leave us in no doubt that hypoxia triggers pulmonary artery constriction by activating mechanisms intrinsic to the smooth muscle and by modulating the release of mediators of constriction/dilation from the pulmonary artery endothelium (Figure 1). Thus pulmonary arterial smooth muscle and endothelial cells are now considered to be representatives of a group of specialized cells, commonly referred to as oxygen-sensing cells, which include carotid body type I cells, neuroepithelial cell bodies found in the airways of the lung and erythropoetin-secreting cells of the kidney. Such cells are defined by their acute sensitivity to relatively small changes in PO2 and have evolved to monitor oxygen supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport oxygen .
Inhibition of mitochondrial oxidative phosphorylation may underpin the regulation by hypoxia of oxygen-sensing cells
The precise mechanism(s) by which hypoxia elicits pulmonary artery constriction represents a more contentious issue. However, comparative studies on different oxygen-sensing cell types, including pulmonary arterial smooth muscle cells, carotid body type I cells and neuroepithelial cell bodies, have provided one consistent finding. That is that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation (for review see ) as indicated by depolarization of the mitochondrial membrane potential and/or by an increase in β-NADH levels, the latter having been shown to occur in isolated pulmonary arteries [17,18]. Such effects are observed over a range of PO2 that elicits no such change in mitochondrial function in cells that do not function to monitor PO2 and led, therefore, to the proposal that the inhibition of mitochondrial oxidative phosphorylation may underpin, at least in part, the activation of oxygen-sensing cells by hypoxia. Others have argued that the affinity of cytochrome c oxidase for oxygen is too high to allow for the inhibition of mitochondrial oxidative phosphorylation by physiological levels of hypoxia. However, there is clear evidence to suggest that respiratory control by oxygen may be determined by cell- and tissue-specific differences in: (i) the metabolic environment of mitochondria; (ii) the oxygen affinity of cytochrome c oxidase; and (iii) the dependence of the oxygen affinity of cytochrome c oxidase on metabolic state and rate .
How may the inhibition of mitochondrial metabolism by hypoxia couple to pulmonary artery constriction?
Despite extensive investigation, the mechanism(s) that may couple inhibition of mitochondrial oxidative phosphorylation to activation of oxygen-sensing cells has remained elusive. With respect to HPV there are currently three discrete hypotheses (Figure 2).
A reduction in the cellular redox status
As mentioned previously, one consequence of the inhibition of mitochondrial oxidative phosphorylation by hypoxia would be a reduction in the cellular redox status. Clearly, β-NADH levels would be expected to increase due to reduced oxidation of β-NADH at complex I of the electron transport chain and by a consequent increase in β-NADH generation via anaerobic glycolysis. Thus hypoxia has been shown to increase β-NADH levels in pulmonary artery lysates and to increase β-NAD(P)H autofluorescence in isolated pulmonary artery rings [17,18]. Furthermore, hypoxia may increase the levels of reduced glutathione. It has been proposed, therefore, that HPV may be mediated by an increase in the levels of reduced cellular redox couples and/or by a reduction in the production of ROS (reactive oxygen species) by mitochondria, with cell signalling in response to hypoxia consequent to the reduction of sulfydryl groups on cysteine and methionine residues within key signalling proteins . It should be noted, however, that although reducing agents (e.g. dithiothrietol) and some mitochondrial inhibitors (e.g. rotenone) may induce pulmonary artery constriction, investigations have yet to demonstrate that a reduced cellular redox status alone may elicit pulmonary artery constriction with the precise characteristics of HPV.
A paradoxical increase in the production of ROS
In marked contrast with the above is the proposal that a paradoxical increase in ROS may be elicited by a fall in oxygen supply. This hypothesis suggests that ROS production occurs at complex III of the mitochondrial electron transport chain, due to the uncoupled movement of electrons across the electron transport chain and their consequent interaction with molecular oxygen to form superoxide. Under normoxic conditions, complex III of the electron transport chain serves to accept electrons from ubiquinol and then transfers them to cytochrome c while translocating protons across the inner mitochondrial membrane. Central to this process is the Q cycle, by which complex III converts paired electron transfer from complex I and II into a sequential electron transfer that is required by complex IV. Ubiquinol, which binds to complex III at the Qo site located at the inner mitochondrial membrane, transfers an electron to the Rieske iron-sulfur protein, for onward transfer to cytochrome c. This process yields the univalently reduced ubisemiquinone, which subsequently transfers a second electron to cytochrome b for onward transfer to a second quinone site (Qi) at the inner mitochondrial membrane. It has been proposed that hypoxia reduces the rate of electron transfer, thereby increasing the half-life of ubisemiquinone, and that this prolonged half-life allows molecular oxygen to capture an electron from ubisemiquinone to yield superoxide even though substrate (oxygen) supply has declined. Subsequent metabolism of superoxide to H2O2 via mitochondrial superoxide dismutase would then increase H2O2 levels sufficiently to allow for diffusion through the cytoplasm and activation, via oxidation of cysteine and methionine residues in key proteins, signalling pathways that underpin HPV . Evidence in support of the view that hypoxia increases ROS production and thereby induces a concomitant increase in intracellular calcium concentration has been presented . However, these studies were performed on cultured pulmonary arterial smooth muscle cells, which are most likely a de-differentiated, non-contractile and proliferative phenotype and not representative of acutely isolated pulmonary artery smooth muscle cells. Furthermore, it should be noted that current techniques for the measurement of cellular ROS are notoriously difficult to perform and have provided conflicting data. Moreover, confirmatory studies in support of a role for ROS in mediating HPV in isolated pulmonary arteries have relied on the use of inhibitors of the mitochondrial electron transport chain  and could be interpreted as demonstrating that prior inhibition of mitochondrial oxidative phosphorylation occludes HPV without consideration of a role for ROS. However, the most significant argument against this hypothesis is the fact that acute hyperoxia, which is generally accepted to increase ROS formation, fails to induce an increase in pulmonary vascular resistance or alter the distribution of blood flow in the lung .
When evaluating the aforementioned hypotheses, however, it is important to consider the full extent of conflicting data now available in each case, which has precluded unification of the field behind either proposal .
The cellular energy status and AMPK (AMP-activated protein kinase)
Several investigations have addressed the possibility that a fall in cellular energy status may underpin HPV . Assessment of the information provided by a variety of techniques has clearly shown that the cellular ATP availability/phosphorylation potential remains remarkably stable during exposure of pulmonary arteries to hypoxia, despite the fact that mitochondrial oxidative phosphorylation is inhibited. However, previous assessment of the role of the cellular energy status may have been limited by knowledge of the identity of the energy variable that might signal the response. Significantly, the role of the AMP/ATP ratio and downstream effectors had not been considered until recently, despite the fact that a variety of metabolic stresses (e.g. hypoxia, anoxia, glucose deprivation and cell activation) will precipitate an increase in the AMP/ATP ratio and that this may occur in the absence of a measurable fall in cellular ATP levels. This is due to the action of adenylate kinase, which serves to maintain cellular ATP supply during the initial phase of metabolic stress. To achieve this, adenylate kinase converts two molecules of ADP into AMP+ATP. Thus any increase in the cellular ADP/ATP ratio is converted into a much larger rise in the cellular AMP/ATP ratio. One major consequence of this is the activation of AMPK.
AMPK is a serine/threonine kinase comprising a catalytic α-subunit and regulatory β- and γ-subunits, and has come to prominence as a metabolic fuel gauge which monitors the cellular AMP/ATP ratio as an index of metabolic stress and initiates compensatory changes in cell metabolism in order to maintain ATP supply. Activation of AMPK in response to metabolic stress is most likely mediated by the binding of AMP to two Bateman (cystathionine β-synthase) domains on the γ-subunit of AMPK, which: (i) confers allosteric regulation via the γ-subunit; (ii) permits phosphorylation of the α-subunit at Thr172 by an upstream kinase that is a complex between the tumour suppressor kinase LKB1 and two accessory proteins STRAD (STE20-related adaptor protein) and MO25 (mouse protein 25); and (iii) inhibits dephosphorylation of AMPK. In the absence of metabolic stress, each of these processes is antagonized by high concentrations of ATP, for which the Bateman domains on the γ-subunit have lower affinity than they do for AMP. Thus AMPK is regulated by a triple mechanism that is exquisitely sensitive to very small changes in the AMP/ATP ratio.
Upon activation, AMPK promotes catabolic pathways in order to maintain ATP supply (e.g. β-oxidation of fatty acids), while switching off non-essential anabolic (ATP-consuming) pathways. Thus the primary targets for AMPK had been presumed to be genes and proteins involved in energy metabolism, particularly lipid and carbohydrate metabolism . However, it is now recognised that AMPK can also target non-metabolic processes. In this respect it is important to note that while AMPK is ubiquitously expressed throughout eukaryotic cells, at least 12 different heterotrimers may be formed from multiple isoforms of the catalytic α-subunit (α1 and α2) and regulatory β- (β1 and β2) and γ- (γ1–3) subunits; splice variants of which may add to the diversity. Thus the selective expression of a particular AMPK isozyme(s) could determine, at least in part, both cell- and system-specific responses to metabolic stresses. It has therefore been proposed that together with a reliance on mitochondrial oxidative phosphorylation for ATP production, the tissue-specific expression of a given AMPK heterotrimeric subunit combination could allow for the selective activation of oxygen-sensing cells in response to physiological levels of hypoxia .
Consistent with a role for AMPK in mediating HPV, the AMP/ATP ratio in pulmonary arterial smooth muscle has now been shown to rise 2-fold in response to hypoxia (16–21 mmHg) and vary approximately as the square of the ADP/ATP ratio, as one would expect if the adenylate kinase reaction remained close to equilibrium. Thus the AMP/ATP ratio in pulmonary artery smooth muscle may increase markedly in response to hypoxia although ATP levels may be maintained. As a consequence AMPK is activated, with catalytic activity associated with the α1 subunit isoform being selectively augmented in the acute phase. This may be significant, because under normoxic conditions the level of catalytic activity associated with the AMPKα1 subunit isoform is inversely related to pulmonary artery diameter, as is the magnitude of HPV, and is 4-fold higher in pulmonary arterial smooth muscle when compared with systemic arterial smooth muscle. Thus a specific AMPKa1-associated heterotrimeric subunit combination may provide, via signal amplification, for a degree of pulmonary selectivity that we know is required for HPV. Most significantly, pharmacological activation of AMPK has now been shown to induce pulmonary artery constriction with the precise characteristics of HPV , while an AMPK antagonist has been shown to inhibit acute HPV in isolated pulmonary arteries .
Regulation of pulmonary arterial smooth muscle by hypoxia: calcium release from the sarcoplasmic reticulum underpins smooth muscle constriction
Electrophysiological investigations have demonstrated that hypoxia inhibits the potassium current carried by Kv (voltage-sensitive potassium) channels in pulmonary arterial smooth muscle. This has led to the suggestion that HPV may be initiated by consequent membrane depolarization and voltage-gated calcium influx. However, it should be noted that although Kv channel currents were inhibited at positive membrane potentials, i.e. outside the physiological range, they were actually augmented within the range of the resting membrane potential . Thus, despite the fact that other potassium channels may be inhibited by hypoxia in the appropriate membrane potential range, it is unlikely that there will be a net reduction in potassium conductance sufficient to elicit depolarization necessary for a significant increase in voltage-gated calcium influx. Despite extensive effort in this area, therefore, a contribution to acute HPV of voltage-gated calcium influx by hypoxia has yet to be established. This may explain why voltage-gated calcium channel antagonists have proved disappointing in the treatment of HPH, where their beneficial effects on pulmonary vascular resistance may be consequent to a reduction in systemic vascular resistance and cardiac output . However, it should be noted that such antagonists represent the therapy of choice for hypoxic pulmonary oedema induced at high-altitude .
Regulation by hypoxia of calcium mobilization from sarcoplasmic reticulum calcium stores in pulmonary artery smooth muscle
Several investigations on isolated pulmonary artery smooth muscle cells and isolated pulmonary arteries have established that Phase 1 of HPV is mediated, at least in part, by the release of calcium from SR (sarcoplasmic reticulum) stores via ryanodine receptors . However, there is now compelling evidence in support of a pivotal role for continued smooth muscle SR calcium release via ryanodine receptors in the maintenance of sustained HPV in isolated pulmonary arteries both with and without endothelium [10,12,30]. Briefly, both Phase 1 and Phase 2 of HPV are abolished following the block of SR calcium release via ryanodine receptors with ryanodine and caffeine [10,12], whilst constriction in response to membrane depolarization (80 mM potassium) and consequent voltage-gated calcium influx remain unaffected. Furthermore, constriction of pulmonary artery rings without the endothelium remains unaffected after removal of extracellular calcium, despite the fact that constriction induced by depolarization (80 mM potassium) is abolished. Thus it would appear that smooth muscle calcium release from a ryanodine-sensitive SR store underpins pulmonary artery smooth muscle constriction by hypoxia, rather than voltage-gated calcium entry. This view is supported by a more recent study  that has demonstrated that block of ryanodine receptors with ryanodine attenuates HPV in the isolated rat lung.
The calcium-mobilizing messenger cADPR (cyclic adenosine diphosphate-ribose) may mediate smooth muscle SR calcium release in response to hypoxia
cADPR is one of a family of calcium-mobilizing pyridine nucleotides and is synthesized and broken down by a multi-functional enzyme named ADP-ribosyl cyclase, which has yet to be characterized at the molecular level. An increase in cellular cADPR levels can, via an intermediate ‘cADPR receptor’ that remains to be identified, either increase the sensitivity of ryanodine receptors to calcium-induced calcium release or mobilize SR calcium stores via ryanodine receptor activation . The fact that cADPR represents an endogenous regulator of ryanodine receptor function raised the possibility that hypoxia may mediate SR calcium release, at least in part, by increasing cADPR synthesis in pulmonary arterial smooth muscle cells.
Consistent with a role for cADPR in HPV, the vascular distribution of ADP-ribosyl cyclase activity was found to offer a degree of pulmonary selectivity, being inversely related to pulmonary artery diameter, as is the magnitude of HPV , and at least an order of magnitude higher in homogenates of pulmonary artery smooth muscle than in those of aortic or mesenteric artery smooth muscle . Furthermore, hypoxia (16–21 mm Hg) increased cADPR levels 2-fold in second order branches of the pulmonary artery, and 10-fold in third order branches . Therefore the differential expression of ADP-ribosyl cyclase may offer, via signal amplification, a degree of pulmonary selectivity that is necessary for HPV. The precise mechanism(s) by which hypoxia elicits cADPR accumulation in pulmonary artery smooth muscle remains to be elucidated. However, there is evidence consistent with the view that the metabolic sensor AMPK may couple, at least in part, inhibition of mitochondrial oxidative phosphorylation to cADPR-dependent SR calcium release . Furthermore, this process may be enhanced by β-NADH formation consequent to inhibition of mitochondrial oxidative phosphorylation, because β-NADH may facilitate cADPR accumulation from β-NAD+ by augmenting ADP-ribosyl cyclase and/or inhibiting cADPR hydrolase activities . It should also be noted, however, that there is evidence to suggest that an increase in cellular ROS may also augment cADPR synthesis . Thus all three mechanisms that have been proposed to couple inhibition of mitochondrial function to HPV may regulate either cADPR accumulation and/or cADPR-dependent SR calcium release.
The most definitive evidence in support of a role for cADPR in mediating SR calcium release in response to hypoxia has been provided by experiments using 8-bromo-cADPR, a membrane permeant cADPR antagonist. Thus, pre-incubation of isolated pulmonary artery rings with and without endothelium, with 8-bromo-cADPR abolished the development of Phase 2 of HPV and abolished HPV in the ventilated and perfused rat lung in situ [12,30]. Thus cADPR-dependent SR calcium release via ryanodine receptors may be a pre-requisite for the full expression of HPV in the whole lung ; it is worth noting that blocking of the endothelium-dependent component of HPV by 8-bromo-cADPR may be indirect, due to the fact that an endothelium-derived vasoconstrictor released by hypoxia may mediate pulmonary artery constriction by sensitizing the contractile apparatus to calcium released from the smooth muscle SR in response to hypoxia (see below).
Surprisingly, however, 8-bromo-cADPR had no effect on the transient Phase 1 constriction of isolated pulmonary arteries, despite the fact that this component of HPV was abolished by blockage of ryanodine receptor function. Therefore it seems unlikely that the Phase 1 constriction observed in isolated pulmonary artery rings contributes to either the initiation or maintenance of the increase in pulmonary vascular perfusion pressure at the level of the whole lung, although there may be an alternate physiological consequence (see below).
Store-depletion activated calcium entry and the maintenance of HPV
Maintained smooth muscle constriction in response to hypoxia exhibits a partial dependence on transmembrane calcium influx. In this respect it is of major significance that blockage of SR calcium release via ryanodine receptors with caffeine and ryanodine, or blockage of cADPR with 8-bromo-cADPR blocks the constriction of pulmonary arteries, with or without endothelium, by hypoxia. Thus the partial dependence of smooth muscle constriction on extracellular calcium is most likely due to calcium influx via the recruitment of an SR calcium release-activated store-refilling current [33,34]. However, this process of store-release activated calcium influx is clearly consequent to the mobilization of SR calcium stores rather than being directly regulated by hypoxia [26,33,34].
Does the process underpinning Phase 1 of HPV in isolated pulmonary arteries have any physiological role?
Clearly, Phase 1 of HPV in isolated pulmonary arteries is a robust response that is dependent on calcium release from the SR via ryanodine receptors. However, the contribution of the underlying mechanisms to the hypoxia-induced increase in perfusion pressure in the whole lung is questionable, so what might its physiological function be? One possibility is that a secondary action of hypoxia may be to selectively deplete a functionally segregated SR calcium store that sits in close apposition to the plasma membrane, and which normally supports vasodilation in response to activation of adenylate cyclase-coupled receptors by releasing calcium proximal to the plasma membrane in order to initiate smooth muscle hyperpolarization via the recruitment of calcium-activated potassium channels . Consistent with this proposal is the finding that β-adrenoceptor agonists have been found to be ineffective in the treatment of HPH, due to the fact that pulmonary vasodilation by this pathway is attenuated by hypoxia .
Regulation of the pulmonary artery endothelium by hypoxia promotes smooth muscle constriction via myofilament calcium sensitization
Removal of the pulmonary arterial endothelium or removal of extracellular calcium attenuates Phase 2 of HPV by abolishing the slow, tonic constriction that develops over ~40 min. Thus calcium influx into pulmonary arterial endothelial cells is likely to underpin the endothelium-dependent component of HPV. This appears to be mediated by a reduction in the bioavailability of endothelium-derived nitric oxide  and the release of an endothelium-derived vasoconstrictor. The nature of the vasoconstrictor remains controversial, although there is evidence to support a role for endothelin-1 . However, more recent studies have suggested that a distinct vasoconstrictor may be the main protagonist . Its nature remains to be determined, but current evidence suggests that it is heat stable and pulmonary selective. Perhaps most significantly, this vasoconstrictor does not appear to mobilize smooth muscle SR calcium stores or trigger voltage-gated calcium influx into pulmonary arterial smooth muscle. Consistent with this finding, the endothelium-dependent component of HPV develops in a manner that is uncoupled from further changes in intracellular calcium concentration in the smooth muscle. Thus it has been proposed that the principle vasoconstrictor released from the endothelium in response to hypoxia increases the sensitivity of the smooth muscle contractile apparatus to calcium .
In smooth muscle, the major determinant of cross-bridge cycling is the activated MLCK (myosin light chain kinase) to activated MLCP (myosin light chain phosphatase) ratio. This determines the level of phosphorylation of the MLCs (myosin light chains) and thereby the degree of contraction of the muscle. Classically, an increase in intracellular calcium concentration leads to calcium-calmodulin-dependent activation of MLCK and thereby increases the activated MLCK/activated MLCP ratio. However, it is now generally recognised that the activated MLCK/activated MLCP ratio may be increased in a calcium-independent manner by phosphorylation and consequent inhibition of MLCP. This process of MLCP phosphorylation can be induced by a number of signalling pathways that are regulated by G-protein-coupled receptors, and in this respect the monomeric G-protein RhoA and Rho-kinase (a coiled-coil-forming serine/threonine kinase) may be of primary importance in arterial smooth muscle .
There is now compelling evidence to support a role for the RhoA/Rho-kinase pathway in calcium sensitization during HPV. Firstly, Y-27632, a potent and highly selective Rho-kinase antagonist, inhibits both HPV in isolated rat pulmonary arteries and the hypoxic pressor response in the rat lung . Most recently, hypoxia has been shown to increase Rho-kinase activity by ~260% in isolated pulmonary arteries . Furthermore, in endothelium-denuded arteries the increase in Rho-kinase in response to hypoxia was much lower (~40%) and of a similar magnitude to that observed in cultured smooth muscle cells . Therefore smooth muscle myofilament calcium sensitization in response to hypoxia is likely to be mediated by Rho-kinase and in an endothelium-dependent manner. It is also evident that Rho-kinase activation may represent a primary driving force for the development of hypoxic pulmonary hypertension, and that Rho-kinase antagonists may be advantageous in the treatment of this condition in humans . To date, however, the complexities of this signalling pathway with respect to HPV remain to be determined. Despite this fact we can gain some insight from studies on other smooth muscle preparations (Figure 3).
RhoA is a member of a subfamily of the small monomeric GTP-binding proteins which act as molecular ‘on-off’ switches controlling a number of cellular processes. Like all Rho proteins, RhoA cycles between active GTP-bound and inactive GDP-bound forms. It is in the GTP-bound form that it mediates cellular processes via interactions with downstream effectors. This cycle is under the control of three groups of regulatory proteins. In the inactive GDP-bound form, RhoA is locked in the cytosol by GDIs (guanine nucleotide dissociation inhibitors), which prevent GTP binding and consequent translocation to the plasma membrane. Upon activation of G-protein-coupled receptors, GEFs (guanine-nucleotide-exchange factors), which catalyse the exchange of GDP for GTP by Rho proteins, are activated. As a consequence GTP–RhoA translocates to the plasma membrane, leading to activation of downstream effector molecules. Subsequently, inactivation of RhoA occurs via its intrinsic GTPase activity, which may be accelerated via the action of GAPs (GTPase-activating proteins).
Rho-kinase is activated when GTP–RhoA binds to its C-terminus, which uncovers a catalytic phosphotransferase domain. Once activated Rho-kinase phosphorylates MLCP at its myosin-binding site. This inhibits the catalytic subunit of MLCP (PP1c) and thereby reduces dephosphorylation of MLC. Thus activation of Rho-kinase results in an increase in the activated MLCK/activated MLCP ratio and thereby sensitizes the contractile apparatus to calcium. There are, however, additional pathways via which Rho-kinase may promote myofilament calcium sensitization, for example: (i) phosphorylation of the protein kinase C substrate CPI-17, a phospho-protein which acts as an inhibitory modulator of MLCP; (ii) direct and calcium-independent phosphorylation of MLCs; and (iii) inhibition of LIM kinase, which disrupts the actin depolymerizing protein cofilin and thereby stabilizes filamentous actin .
• Current evidence suggests that HPV is initiated in a manner independent of the autonomic nervous system, but dependent upon the intrinsic response of pulmonary arterial smooth muscle and endothelial cells to hypoxia.
• Activation of each of these cell types occurs, at least in part, as a consequence of the PO2-dependent inhibition of mitochondrial oxidative phosphorylation.
• This may lead to the activation of AMPK combined with an increase in cellular β-NADH levels, or alternatively to a paradoxical increase in ROS production by mitochondria.
• Thereafter cADPR-dependent mobilization of calcium from the smooth muscle SR via ryanodine receptors is induced, together with calcium influx due to consequent activation of an SR store-refilling current. Thus pulmonary artery constriction will be initiated and maintained via calcium/calmodulin-dependent activation of MLCK.
• Pulmonary artery constriction may then be augmented by (i) reduced nitric oxide bioavailability; and (ii) the release of a vasoconstrictor from the pulmonary artery endothelium which may elicit smooth muscle myofilament calcium sensitization via Rho-kinase activation, leading to inhibition of MLCP and a consequent increase in the activated MLCK/activated MLCP ratio.
- © The Authors Journal compilation © 2007 Biochemical Society