Gavin D. Thomas

Mechanisms of the prostaglandin F2α-induced rise in [Ca2+]i in rat intrapulmonary arteries

The mechanisms by which prostaglandin F2α (PGF2α) increases intracellular Ca2+ concentration [Ca2+]i in vascular smooth muscle remain unclear. We examined the role of store‐, receptor‐ and voltage‐operated Ca2+ influx pathways in rat intrapulmonary arteries (IPA) loaded with Fura PE‐3. Low concentrations (0.01–1 μm) of PGF2α caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 μm PGF2α. At higher concentrations of PGF2α, a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 μm PGF2α was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 μm PGF2α was mimicked by the TP receptor agonist U‐46619. The plateau rise in [Ca2+]i in response to 0.1 μm PGF2α was insensitive to diltiazem, and was abolished in Ca2+‐free physiological salt solution, and by pretreatment with La3+, 2‐APB, thapsigargin or U‐73122. The rises in [Ca2+]i in response to 10 μm PGF2α and 0.01 μm U‐46619 were partially inhibited by diltiazem. The diltiazem‐resistant components of both of these responses were inhibited by 2‐APB and La3+ to an extent which was significantly less than that seen for the response to 0.1 μm PGF2α, and were also much less sensitive to U‐73122. The U‐46619 response was also relatively insensitive to thapsigargin. When Ca2+ was replaced with Sr2+, the sustained increase in the Fura PE‐3 signal to 0.1 μm PGF2α was abolished, whereas 10 μm PGF2α and 0.05 μm U‐46619 still caused substantial increases. These results suggest that low concentrations of PGF2α act via FP receptors to cause IP3‐dependent Ca2+ release and store operated Ca2+ entry (SOCE). U‐46619 and 10–100 μm PGF2α cause a TP receptor‐mediated Ca2+ influx involving both L‐type Ca2+ channels and a receptor operated pathway, which differs from SOCE in its susceptibility to La3+, 2‐APB and thapsigargin, does not require phospholipase C activation, and is Sr2+ permeable.

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.

Mechanisms of PGF2α induced rises in [Ca2+]i in rat intrapulmonary arteries

The mechanisms by which prostaglandin F2α (PGF2α) increases intracellular Ca2+ concentration [Ca2+]i in vascular smooth muscle remain unclear. We examined the role of store‐, receptor‐ and voltage‐operated Ca2+ influx pathways in rat intrapulmonary arteries (IPA) loaded with Fura PE‐3. Low concentrations (0.01–1 μm) of PGF2α caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 μm PGF2α. At higher concentrations of PGF2α, a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 μm PGF2α was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 μm PGF2α was mimicked by the TP receptor agonist U‐46619. The plateau rise in [Ca2+]i in response to 0.1 μm PGF2α was insensitive to diltiazem, and was abolished in Ca2+‐free physiological salt solution, and by pretreatment with La3+, 2‐APB, thapsigargin or U‐73122. The rises in [Ca2+]i in response to 10 μm PGF2α and 0.01 μm U‐46619 were partially inhibited by diltiazem. The diltiazem‐resistant components of both of these responses were inhibited by 2‐APB and La3+ to an extent which was significantly less than that seen for the response to 0.1 μm PGF2α, and were also much less sensitive to U‐73122. The U‐46619 response was also relatively insensitive to thapsigargin. When Ca2+ was replaced with Sr2+, the sustained increase in the Fura PE‐3 signal to 0.1 μm PGF2α was abolished, whereas 10 μm PGF2α and 0.05 μm U‐46619 still caused substantial increases. These results suggest that low concentrations of PGF2α act via FP receptors to cause IP3‐dependent Ca2+ release and store operated Ca2+ entry (SOCE). U‐46619 and 10–100 μm PGF2α cause a TP receptor‐mediated Ca2+ influx involving both L‐type Ca2+ channels and a receptor operated pathway, which differs from SOCE in its susceptibility to La3+, 2‐APB and thapsigargin, does not require phospholipase C activation, and is Sr2+ permeable.

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.

Mechanisms of the prostaglandin F2α-induced rise in [Ca2+]iin rat intrapulmonary arteries: PGF2αand [Ca2+]iin intrapulmonary arteries

The mechanisms by which prostaglandin F2α (PGF2α) increases intracellular Ca2+ concentration [Ca2+]i in vascular smooth muscle remain unclear. We examined the role of store‐, receptor‐ and voltage‐operated Ca2+ influx pathways in rat intrapulmonary arteries (IPA) loaded with Fura PE‐3. Low concentrations (0.01–1 μm) of PGF2α caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 μm PGF2α. At higher concentrations of PGF2α, a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 μm PGF2α was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 μm PGF2α was mimicked by the TP receptor agonist U‐46619. The plateau rise in [Ca2+]i in response to 0.1 μm PGF2α was insensitive to diltiazem, and was abolished in Ca2+‐free physiological salt solution, and by pretreatment with La3+, 2‐APB, thapsigargin or U‐73122. The rises in [Ca2+]i in response to 10 μm PGF2α and 0.01 μm U‐46619 were partially inhibited by diltiazem. The diltiazem‐resistant components of both of these responses were inhibited by 2‐APB and La3+ to an extent which was significantly less than that seen for the response to 0.1 μm PGF2α, and were also much less sensitive to U‐73122. The U‐46619 response was also relatively insensitive to thapsigargin. When Ca2+ was replaced with Sr2+, the sustained increase in the Fura PE‐3 signal to 0.1 μm PGF2α was abolished, whereas 10 μm PGF2α and 0.05 μm U‐46619 still caused substantial increases. These results suggest that low concentrations of PGF2α act via FP receptors to cause IP3‐dependent Ca2+ release and store operated Ca2+ entry (SOCE). U‐46619 and 10–100 μm PGF2α cause a TP receptor‐mediated Ca2+ influx involving both L‐type Ca2+ channels and a receptor operated pathway, which differs from SOCE in its susceptibility to La3+, 2‐APB and thapsigargin, does not require phospholipase C activation, and is Sr2+ permeable.