Tom P. Robertson

Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat

1 It has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated via K+ channel inhibition and Ca2+ influx through voltage‐gated channels. HPV depends strongly on the degree of preconstriction, and we therefore examined the effect of Ca2+ channel blockade on tension and intracellular [Ca2+] ([Ca2+]i) during HPV in rat intrapulmonary arteries (IPAs), whilst maintaining preconstriction constant. We also investigated the role of intracellular Ca2+ stores. 2 HPV demonstrated a transient constriction (phase I) superimposed on a sustained constriction (phase II). Nifedipine (1 μm) partially inhibited phase I, but did not affect phase II. In arteries exposed to 80 mm K+ and nifedipine or diltiazem the rises in tension and [Ca2+]i were blunted during phase I, but were unaffected during phase II. 3 At low concentrations (< 3 μm), La3+ almost abolished the phase I constriction and rise in [Ca2+]i, but had no effect on phase II, or constriction in response to 80 mm K+. Phase II was inhibited by higher concentrations of La3+ (IC50∼50 μm). 4 IPA treated with thapsigargin (1 μm) in Ca2+‐free solution to deplete Ca2+ stores showed sustained constriction upon re‐exposure to Ca2+ and an increase in the rate of Mn2+ influx, suggesting capacitative Ca2+ entry. The concentration dependency of the block of constriction by La3+ was similar to that for phase I of HPV. Pretreatment of IPA with 30 μm CPA reduced phase I by > 80%, but had no significant effect on phase II. 5 We conclude that depolarization‐mediated Ca2+ influx plays at best a minor role in the transient phase I constriction of HPV, and is not involved in the sustained phase II constriction. Instead, phase I appears to be mainly dependent on capacitative Ca2+ entry related to release of thapsigargin‐sensitive Ca2+ stores, whereas phase II is supported by Ca2+ entry via a separate voltage‐independent pathway.

Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat

We have examined the effects of Y‐27632, a specific inhibitor of Rho‐activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y‐27632 (100 nM–3 μM) was found to cause a concentration‐dependent inhibition of acute sustained HPV in rat IPA. Application of Y‐27632 (10–600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration‐dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y‐27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho‐associated kinase may be a pivotal step in the generation of sustained HPV.

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 Ca2+ ([Ca2+]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 μm) 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 [Ca2+]i during HPV. 4 Depolarisation‐induced constriction was unaffected by hypoxia except in the absence of glucose, when it was depressed by ∼50 %. Depolarisation‐induced constriction was depressed by rotenone during hypoxia by 23 ± 4 %; cyanide was without effect. 5 Hypoxia increased 2‐deoxy‐[3H]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 [Ca2+]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 Ca2+‐sensitisation pathway rather than the rise in [Ca2+]i.

Hypoxic pulmonary vasoconstriction: mechanisms and controversies

The pulmonary circulation differs from the systemic in several important aspects, the most important being that pulmonary arteries constrict to moderate physiological (∼20–60 mmHg PO2) hypoxia, whereas systemic arteries vasodilate. This phenomenon is called hypoxic pulmonary vasoconstriction (HPV), and is responsible for maintaining the ventilation–perfusion ratio during localized alveolar hypoxia. In disease, however, global hypoxia results in a detrimental increase in total pulmonary vascular resistance, and increased load on the right heart. Despite many years of study, the precise mechanisms underlying HPV remain unresolved. However, as we argue below, there is now overwhelming evidence that hypoxia can stimulate several pathways leading to a rise in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMC). This rise in [Ca2+]i is consistently found to be relatively small, and HPV seems also to require rho kinase‐mediated Ca2+ sensitization. There is good evidence that HPV also has an as yet unexplained endothelium dependency. In this brief review, we highlight selected recent findings and ongoing controversies which continue to animate the study of this remarkable and unique response of the pulmonary vasculature to hypoxia.

Endothelium-derived mediators and hypoxic pulmonary vasoconstriction

The vascular endothelium synthesises, metabolises or converts a multitude of vasoactive mediators, and plays a vital role in the regulation of pulmonary vascular resistance. Its role in hypoxic pulmonary vasoconstriction (HPV) is however controversial. Although HPV has been demonstrated in both pulmonary arteries where the endothelium has been removed and isolated pulmonary artery smooth muscle cells, many reports have shown either partial or complete dependence on an intact endothelium for sustained HPV (> approximately 20 min). However, despite many years of study no known endothelium-derived mediator has yet been unequivocally shown to be essential for HPV, although several may either facilitate the response or act as physiological brakes to limit the extent of HPV. In this article we review the evidence for and against the role of specific endothelium-derived mediators in HPV. We make the case for a facilitatory or permissive function of the endothelium, that in conjunction with a rise in smooth muscle intracellular Ca(2+) initiated by a mechanism intrinsic to smooth muscle, allows the development of sustained HPV. In particular, we propose that in response to hypoxia the pulmonary vascular endothelium releases an as yet unidentified agent that causes Ca(2+) sensitisation in the smooth muscle.

Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat

The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200–500 μm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined. Following incubation of IPA with 100 nM thapsigargin (Thg) in Ca2+‐free physiological salt solution (PSS), a sustained contraction was observed upon reintroduction of 1.8 mM Ca2+, which was unaffected by either diltiazem (10 μM) or the reverse mode Na+/Ca2+ antiport inhibitor KB‐R7943 (10 μM). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA. The effect of this protocol on the intracellular Ca2+ concentration ([Ca2+]i) was assessed using Fura PE3‐loaded IPA, MA, and FA. Reintroduction of Ca2+ into the bath solution following Thg treatment in Ca2+‐free PSS caused a large, rapid, and sustained increase in [Ca2+]i in all the three types of artery. 100 nM Thg induced a slowly developing noisy inward current in smooth muscle cells (SMC) isolated from IPA, which was due to an increase in the activity of single channels with a conductance of ∼30 pS. The current had a reversal potential near 0 mV in normal PSS, and persisted when Ca2+‐dependent K+ and Cl− currents were blocked; it was greatly inhibited by 1 μM La3+, 1 μM Gd3+, and the IP3 receptor antagonist 2‐APB (75 μM), and by replacement of extracellular cations by NMDG+. In conclusion, depletion of intracellular Ca2+ stores with Thg caused capacitative Ca2+ entry in rat small muscular IPA, MA, and FA. However, a corresponding contraction was observed only in IPA. CCE in IPA was associated with the development of a small La3+‐ and Gd3+‐sensitive current, and an increased Mn2+ quench of Fura PE‐3 fluorescence. These results suggest that although CCE occurs in a number of types of small arteries, its coupling to contraction appears to be of particular importance in pulmonary arteries.

Hypoxia induces the release of a pulmonary-selective, Ca2+-sensitising, vasoconstrictor from the perfused rat lung

OBJECTIVE Sustained hypoxic pulmonary vasoconstriction is dependent upon the presence of an intact endothelium, strongly suggesting that an endothelium-derived constrictor factor is involved in this response. In the present study we have attempted to determine whether hypoxia induces the release of a vasoconstrictor(s) from the lung, and whether this vasoconstrictor shares mechanistic features with the hypoxic constrictor response. METHODS The salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat intrapulmonary artery functional bioassay were utilised in this study. RESULTS Hypoxic, but not normoxic, perfusion of the isolated lung of the rat induced the release of a vasoconstrictor(s) which appeared to be selective for pulmonary over mesenteric arteries of the rat. The vasoconstriction observed was unaffected by inhibition of voltage-gated Ca(2+) channels, and was not associated with a rise in intracellular [Ca(2+)], suggesting Ca(2+)-sensitisation of the contractile apparatus. The vasoconstriction was also unaffected by the protein kinase C (PKC) inhibitor Ro-31-8220, or the endothelin-1 antagonists BQ123/BQ788 but was markedly potentiated in the presence of prostaglandin F(2alpha). CONCLUSION We conclude that hypoxic perfusion of the rat lung results in the release of a vasoconstrictor(s) which shares some of the facets of the sustained hypoxic constriction of isolated intrapulmonary arteries of the rat, since it involves PKC-independent Ca(2+) sensitisation, is independent of voltage-gated Ca(2+) entry, and is potentiated by the presence of preconstriction.

AMP-activated protein kinase and hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction is a vital homeostatic mechanism that aids ventilation-perfusion matching in the lung, for which the underlying mechanism(s) remains controversial. However, our most recent investigations strongly suggest that hypoxic pulmonary vasoconstriction is precipitated, at least in part, by the inhibition of mitochondrial oxidative phosphorylation by hypoxia, an increase in the AMP/ATP ratio and consequent activation of AMP-activated protein kinase (AMPK). Unfortunately, these studies lacked the definitive proof that can only be provided by selectively blocking AMPK-dependent signalling cascades. The aim of the present study was, therefore, to determine the effects of the AMPK inhibitor compound C upon: (1) phosphorylation in response to hypoxia of a classical AMPK substrate, acetyl CoA carboxylase, in rat pulmonary arterial smooth muscle and (2) hypoxic pulmonary vasoconstriction in rat isolated intrapulmonary arteries. Acetyl CoA carboxylase phosphorylation was increased approximately 3 fold in the presence of hypoxia (pO(2) = 16-21 mm Hg, 1 h) and 5-aminoimidazole-4-carboxamide riboside (AICAR; 1 mM; 4 h) and in a manner that was significantly attenuated by the AMPK antagonist compound C (40 microM). Most importantly, pre-incubation of intrapulmonary arteries with compound C (40 microM) inhibited phase II, but not phase I, of hypoxic pulmonary vasoconstriction. Likewise, compound C (40 microM) inhibited constriction by AICAR (1 mM). The results of the present study are consistent with the activation of AMPK being a key event in the initiation of the contractile response of pulmonary arteries to acute hypoxia.

Thromboxane and isoprostanes as inflammatory and vasoactive mediators in black walnut heartwood extract induced equine laminitis

Inflammation and vascular dysfunction occur concurrently during the prodromal stages of equine laminitis. The aim of this study was to provide insights into the role that thromboxane and isoprostanes may play in the development of black walnut heartwood extract (BWHE)-induced laminitis. Horses were divided into two groups, either control or BWHE-administered horses. Plasma concentrations of thromboxane increased transiently after administration of BWHE and coincided with the nadir in white blood cell counts, whereas plasma concentrations of iso-prostaglandin PGF(2alpha) (iso-PGF(2alpha)) did not change in either group. At 12h (for the control group) or Obel grade 1 laminitis (for the BWHE group) the horses were euthanized and laminar tissue collected. Laminar arteries and veins were used in functional studies with vasoconstrictor substances and tissue samples were used for the determination of laminar iso-PGF(2alpha) concentrations. Laminar tissue concentrations of iso-PGF(2alpha) were significantly greater in BWHE horses when compared to control horses. In parallel studies concentrations of iso-PGF(2alpha) in laminar tissue samples obtained 1.5 and 3h after administration of BWHE were indistinguishable from those for control horses at 3 or 12h after administration of an equal volume of water. Laminar vessel constrictor responses to either a thromboxane mimetic (U46619), iso-prostaglandin PGE(2) (iso-PGE(2)) or iso-PGF(2alpha) were determined using small vessel myographs. In some vessels, the effects of putative prostanoid and thromboxane receptor antagonists, SQ 29,548, SC-19220 and AH 6809, upon contractile responses were determined. In control horses, U46619, iso-PGF(2alpha) and iso-PGE(2) more potently and efficaciously constricted laminar veins when compared to laminar arteries. Responses of laminar veins from BWHE horses to iso-PGE(2) were similar to those of laminar veins from control horses, whereas iso-PGF(2alpha) elicited significantly greater responses in laminar veins from BWHE horses when compared to controls. In contrast, responses to U46619 were smaller in laminar veins isolated from BWHE horses when compared to those in laminar veins from control horses. In the presence of SQ 29,548, iso-PGF(2alpha) elicited a small dilation in laminar veins from control horses, which was not apparent in laminar veins from BWHE horses. These results are consistent with both systemic and local inflammatory events occurring during the prodromal stages of BWHE-induced laminitis. Because laminar veins are sensitive to thromboxane and isoprostanes, these substances may act as conduits between the inflammatory and vascular events occurring in laminitis and may be therapeutic targets for this crippling condition.

Equine laminitis: A journey to the dark side of venous

Equine laminitis is a crippling condition that continues to defy repeated efforts to delineate the precise mechanisms involved and develop effective therapeutic strategies for use in the clinic. In this article, the possible role of dysfunction of the laminar vasculature is discussed, with particular emphasis on the venous side of the laminar microvasculature and the possible role(s) that metabolic syndrome and thrombosis may play in the dysfunction observed in the laminar microvasculature during the development of laminitis.