Richard M. Leach

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.

Hypoxia, energy state and pulmonary vasomotor tone

Vasomotor responses to hypoxia constitute a fundamental adaptation to a commonly encountered stress. It has long been suspected that changes in cellular energetics may modulate both hypoxic systemic artery vasodilatation (HSV) and hypoxic pulmonary artery vasoconstriction (HPV). Although limitation of energy has been shown to underlie hypoxic relaxation in some smooth muscles, the response to hypoxia in vascular smooth muscle does not appear to be a simple function of energy stores, but instead may involve perturbations of ATP or energy delivery to mechanisms controlling muscle force, and/or changes associated with anaerobic metabolism. Recent work in pulmonary vascular smooth muscle has demonstrated that energy stores are maintained during hypoxic pulmonary vasoconstriction, and that this is dependent on glucose availability and up-regulation of glycolysis. There is increasing evidence that glycolysis is preferentially coupled to a variety of membrane associated ATP dependent processes, including the Na(+) pump, Ca(2+)-ATPase, and possibly some protein kinases. These and other mechanisms may influence excitation-contraction coupling in both systemic and pulmonary arteries by effects on intracellular Ca(2+) and/or Ca(2+) sensitivity. Hypoxia has also been postulated to have major effects on other cytosolic second messenger systems including phosphatidylinositol pathways, cell redox state and mitochondrial reactive oxygen species production. This review examines the relationship between energy state, anaerobic respiration and hypoxic vasomotor tone, with a particular emphasis on hypoxic pulmonary vasoconstriction.

Low Concentrations of Sphingosylphosphorylcholine Enhance Pulmonary Artery Vasoreactivity The Role of Protein Kinase Cδ and Ca2+ Entry

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC50 is ≈100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (≤1 &mgr;mol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca2+]. SPC (1 &mgr;mol/L) had no effect on force or intracellular [Ca2+] on its own, but dramatically potentiated constrictions induced by ≈25 mmol/L [K+], such that at 40 minutes, force and intracellular [Ca2+] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca2+ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC&dgr; inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC&dgr;. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2&agr; and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 &mgr;mol/L) caused translocation of PKC&dgr; to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca2+ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC&dgr;. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity - The role of protein kinase C delta and Ca2+ entry

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC50 is ≈100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (≤1 &mgr;mol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca2+]. SPC (1 &mgr;mol/L) had no effect on force or intracellular [Ca2+] on its own, but dramatically potentiated constrictions induced by ≈25 mmol/L [K+], such that at 40 minutes, force and intracellular [Ca2+] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca2+ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC&dgr; inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC&dgr;. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2&agr; and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 &mgr;mol/L) caused translocation of PKC&dgr; to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca2+ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC&dgr;. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Low Concentrations of Sphingosylphosphorylcholine Enhance Pulmonary Artery Vasoreactivity

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC50 is ≈100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (≤1 &mgr;mol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca2+]. SPC (1 &mgr;mol/L) had no effect on force or intracellular [Ca2+] on its own, but dramatically potentiated constrictions induced by ≈25 mmol/L [K+], such that at 40 minutes, force and intracellular [Ca2+] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca2+ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC&dgr; inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC&dgr;. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2&agr; and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 &mgr;mol/L) caused translocation of PKC&dgr; to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca2+ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC&dgr;. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

LOW CONCENTRATIONS OF SPHINGOSYLPHOSPHORYLCHOLINE ENHANCE PULMONARY ARTERY VASOREACTIVITY: ROLE OF PKCδ AND Ca2+ ENTRY

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC50 is ≈100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (≤1 &mgr;mol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca2+]. SPC (1 &mgr;mol/L) had no effect on force or intracellular [Ca2+] on its own, but dramatically potentiated constrictions induced by ≈25 mmol/L [K+], such that at 40 minutes, force and intracellular [Ca2+] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca2+ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC&dgr; inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC&dgr;. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2&agr; and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 &mgr;mol/L) caused translocation of PKC&dgr; to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca2+ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC&dgr;. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.