Alan R. Conant

Phenoxybenzamine treatment is insufficient to prevent spasm in the radial artery: the effect of other vasodilators

OBJECTIVES After its reintroduction as an arterial graft in coronary artery surgery, the radial artery is now established as an alternative arterial conduit, with good early and midterm patency. However, because of the concern about its vasospasticity, numerous vasodilator strategies have been used. Recently the use of the irreversible alpha-adrenergic antagonist phenoxybenzamine has been proposed. Although this treatment is effective in eliminating the vasoconstriction mediated by noradrenaline, the contribution of other circulating vasoconstrictors to vasospasm could be as important. This study investigates the response of radial arteries treated with phenoxybenzamine to vasoconstrictor stimuli and possible preventative strategies. METHODS In vitro, sections of radial artery, pretreated with phenoxybenzamine after harvesting, were stimulated with maximal concentrations of the vasoconstrictors noradrenaline, vasopressin, angiotensin II, KCl, and endothelin-1. In matched segments of artery, vasoconstrictor responses were recorded in the presence of diltiazem, glyceryl trinitrate, and papaverine and compared with phenoxybenzamine-treated samples. RESULTS Phenoxybenzamine-treated radial artery failed to respond to noradrenaline but did respond to vasopressin, angiotensin II, endothelin-1, and KCl. Diltiazem was largely ineffective against contractile stimuli apart from KCl. Glyceryl trinitrate and papaverine significantly reduced responses to all of the vasoconstrictors tested. CONCLUSION In phenoxybenzamine-treated sections of radial artery, circulating vasoconstrictor agonists may still contribute to the induction of spasm. Additional vasodilator strategies may be required to completely prevent vasospasm.

cAMP inhibits migration, ruffling and paxillin accumulation in focal adhesions of pancreatic ductal adenocarcinoma cells: effects of PKA and EPAC.

We demonstrated that increasing intracellular cAMP concentrations result in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation is mediated by PKA, whilst EPAC potentiates migration.

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

1 To characterize the P2 receptors present on the human umbilical vein endothelial‐derived cell line, ECV304, cytosolic Ca2+, ([Ca2+]c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists. 2 Concentration response curves were obtained in fura‐2‐loaded ECV304 cell suspensions, with EC50 values of 4.2 μM for ATP, 2.5 μM for UTP and 14 μM for adenosine‐5′‐O‐(3‐thio)triphosphate (ATPγS). EC50 values for 2‐methylthioATP, ADP, adenosine‐5′‐O‐(2‐thio)diphosphate (ADPβS) and AMP were 0.5 μM, 3.5 μM, 15 μM and 4.7 μM respectively, but maximal [Ca2+]c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and β,γ,methyleneATP. 3 Cross‐desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP‐sensitive receptor and AMP recognized a third distinct receptor. 4 ECV304 [Ca2+]c responses to 2‐methylthioATP were inhibited in the presence of 30 μM pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), whereas [Ca2+]c responses to UTP were unaffected by this treatment. 5 ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in [Ca2+]c. Apparent responses to Ap4A, Ap5A and Ap6A, were shown to be due to a minor nucleotide contaminant that could be removed by pre‐treatment of the diadenosine samples with either alkaline phosphatase or apyrase. 6 ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A. In addition, ECV304 cells respond to AMP with increases in [Ca2+]c via an as yet uncharacterized receptor.

Endothelin receptors in cultured and native human radial artery smooth muscle

In human vascular smooth muscle cells endothelin-1, acting at both endothelin A and endothelin B receptors, has been demonstrated to be both a potent vasoconstrictor and mitogen. Our aim was to study the functional expression of endothelin receptors in human radial artery smooth muscle using both native tissue and cultured cells (RASMCs). Radial artery smooth muscle cells were cultured from arterial explants and loaded with the calcium fluorescent dye fura-2. Cells responded to endothelin-1 and a variety of other vasoconstrictors with rises in cytoplasmic calcium ([Ca 2+ ] c ). Arterial rings responded to endothelin-1 with an increase in tension. The response of both cells and arterial rings to endothelin-1 was characterized using the selective endothelin A receptor antagonist BQ123 and the endothelin B receptor antagonist BQ788. The RASMCs were found to express [Ca 2+ ] c responses consistent with the expression of only the endothelin A receptor. Endothelin-1-mediated vasoconstriction in radial artery rings was unaffected by BQ788 but was completely blocked by BQ123. Using the selective radioligands [ 125 I]-PD151242 and [ 125 I]-BQ3020 and a combination of in vitro receptor autoradiography and isolated cell preparations, endothelin A receptors were confirmed to be present on RASMCs and on arterial sections, whereas endothelin B binding was barely detectable on native smooth muscle and on RASMCs.

Diadenosine polyphosphates are largely ineffective as agonists at natively expressed P2Y(1) and P2Y(2) receptors on cultured human saphenous vein endothelial cells.

The diadenosine polyphosphates are a group of long-lasting compounds, released into the bloodstream by platelet degranulation. They mediate endothelium-dependent vasodilatation in several animal vascular systems via P2Y receptors coupled to increases in cytoplasmic calcium ([Ca²⁺]_c). However, there is little evidence of diadenosine-mediated vasodilatation in the human vasculature, and a direct interaction with natively expressed P2Y receptors on human endothelium has not been demonstrated. We have therefore studied the effects of diadenosines on primary cultures of human saphenous vein endothelial cells (HSVECs) and related this to the expression of P2Y receptors. HSVECs were loaded with the calcium-sensitive dye fura-2, and nucleotide-stimulated [Ca²⁺]_c responses were recorded. HSVECs responded to 10 µM UTP, ATP and 2-methylthio-ATP but not to UDP. Consistent with the recorded [Ca²⁺]_c responses, RT-PCR analysis of HSVEC RNA amplified specific products for the P2Y₁ and P2Y₂ receptors but not the P2Y₄ and P2Y₆ receptors. HSVECs responded to Ap₃A with a rise in [Ca²⁺]_c, but none of the other diadenosines tested elicited a response. Therefore natively expressed human P2Y₁ and P2Y₂ receptors are insensitive to diadenosine polyphosphates with the exception of Ap₃A. We would therefore predict that the diadenosine polyphosphates have only a limited vasodilatory role in human saphenous veins.

Attenuation of receptor-dependent and -independent vasoconstriction in the human radial artery

BACKGROUND Vasodilator strategies used to treat bypass grafts in the operating theatre, such as nitrates, phosphodiesterase inhibitors and calcium channel antagonists have a broad but short-lived effect against a variety of vasoconstrictor stimuli. Treatments that react irreversibly with proteins modulating vasoconstriction have the advantage that their effects can last well into the postoperative period. In addition systemic effects are avoided as the treatment is localised to the treated graft. This study investigated the use of two clinically applied drugs; fluphenazine (SKF7171A, HCl), an irreversible calmodulin antagonist and minoxidil sulphate, an irreversible potassium channel opener. Treatments were tested against receptor and non-receptor-mediated contraction in the human radial artery. METHOD Isometric tension was measured in response to angiotensin II, KCl and vasopressin in 108 radial artery rings (taken from 31 patients undergoing coronary artery bypass grafting). Control responses were compared with rings pretreated with fluphenazine or minoxidil sulphate. Vasopressin responses were also compared in the presence of glyceryl trinitrate or the reversible Rho kinase inhibitor Y27632. RESULTS Fluphenazine pretreatment significantly suppressed vasoconstriction to all agonists tested. Maximal responses to angiotensin II, vasopressin and KCl were reduced by 42+/-19%, 35+/-8% and 48+/-15% respectively, without any measurable effect on the EC(50). Minoxidil sulphate showed no discernable effect. Vasopressin-induced contraction was also reduced by high levels of glyceryl trinitrate (220 microM; 50 microg/ml) or 10 microM Y27632. CONCLUSIONS The irreversible calmodulin antagonist fluphenazine has potential to be developed as an inhibitor of contraction in arterial graft vessels. The involvement of Rho kinase indicates that other vasoconstrictors and surgical stress can sensitize radial artery to vasopressin-induced contraction. Strategies targeting this pathway also have future potential.

Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage

Ca2+ is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca2+ signalling and homeostasis. Downstream effects of Ca2+ signalling have been and continue to be an important research avenue. The primary functions regulated by Ca2+ in pancreatic acinar cells – exocytotic secretion and fluid secretion – have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca2+ in cellular pathology and the related question of the interplay between Ca2+ signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review.

Phenoxybenzamine treatment can lead to loss of endothelial cell viability

Phenoxybenzamine, an irreversible alpha-adrenoceptor antagonist, is used as a topical treatment against catecholamine-induced contraction in radial artery bypass grafts. Published data suggest that a wide range of phenoxybenzamine doses may be equally effective. This study aimed to investigate whether lower doses of phenoxybenzamine would benefit grafts by better preserving endothelium. To this end human vascular endothelial cells were isolated from sections of radial artery or saphenous vein, and treated with phenoxybenzamine for 30 min. Cells were then washed free of drug and viability assayed using a resazurin-based toxicology assay or returned to culture for assay at 24 h. Phenoxybenzamine treatment showed a dose-dependent effect on cell viability over several clinically employed concentrations. Concentrations above 0.1 mM led to a loss of viability, which became more pronounced with time. The loss of viability was shown to be independent of the carrier used, as results were identical when phenoxybenzamine was dissolved in dimethylsulphoxide, which alone did not affect viability. Changes in pH alone were also not sufficient to affect viability. In conclusion, phenoxybenzamine treatment is likely to cause damage to graft endothelium if employed at concentrations above 0.1 mM (0.03 mg/ml). Phenoxybenzamine may be safely used at lower doses with no potential loss of endothelial cell viability.

Awards of the ESM

This award entails a donation of 2,500 € to allow younger researchers in the area of microcirculatory and vascular biology research to visit research laboratories to learn new techniques. Personal applications as well as suggestions are invited. The candidate should provide a twopage curriculum vitae including a short section on scientific development and future perspectives. The top ranking applications will be rewarded by a free subscription of the Journal of Vascular Research for two years. Van Leeuwenhoek distinctive Travel Award 2000: Hans Vink (Amsterdam, The Netherlands) for his pioneering studies on the endothelial surface layer