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.

Human erythrocytes parasitized with the malarial protozoan Plasmodium falciparum showed rates of L-lactate, D-lactate, and pyruvate uptake many fold greater than control cells. Thus it was necessary to work at 0 degrees C to resolve true initial rates of transport. Studies on the dependence of the rate of transport on substrate concentration implied the presence in parasitized cells of both a saturable mechanism blocked by alpha-cyano-4-hydroxycinnamate (CHC) and a nonsaturable mechanism insensitive to CHC. The former was dominant at physiological substrate concentrations with Km values for pyruvate and D-lactate of 2.3 and 5.2 mM, respectively, with no stereoselectivity for L- over D-lactate. CHC was significantly less effective as an inhibitor of lactate transport in parasitized erythrocytes than in uninfected cells, whereas p-chloromercuribenzenesulfonate, a potent inhibitor in control cells, gave little or no inhibition of lactate transport into parasitized erythrocytes. Inhibition of transport into infected cells was also observed with phloretin, furosemide, niflumic acid, stilbenedisulfonate derivatives, and 5-nitro-2-(3-phenylpropylamino)benzoic acid at concentrations similar to those that inhibit the lactate carrier of control erythrocytes. These compounds were more effective inhibitors of the rapid transport of chloride into infected cells than of lactate transport, whereas CHC was more effective against lactate transport. This implies that different pathways are involved in the parasite-induced transport pathways for lactate and chloride. The transport of L-lactate into infected erythrocytes was also inhibited by D-lactate, pyruvate, 2-oxobutyrate, and 2-hydroxybutyrate. The intracellular accumulation of L-lactate at equilibrium was dependent on the transmembrane pH gradient, suggesting a protogenic transport mechanism. Our data are consistent with lactate and pyruvate having direct access to the malarial parasite, perhaps via the proposed parasitophorous duct or some close contact between the host cell and parasite plasma membranes, with transport across the latter by both a proton-linked carrier (CHC-sensitive, saturable, and the major route) and free diffusion of the undissociated acid (CHC-insensitive, unsaturable, and a minor route).

1 To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca 2+ , ([Ca 2+ ] 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 EC 50 values of 4.2 mM for ATP, 2.5 mM for UTP and 14 mM for adenosine-5'-O-(3-thio)triphosphate (ATPgS). EC 50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADPbS) and AMP were 0.5 mM, 3.5 mM, 15 mM and 4.7 mM respectively, but maximal [Ca 2+ ] c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and b,g,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 [Ca 2+ ] c responses to 2-methylthioATP were inhibited in the presence of 30 mM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whereas [Ca 2+ ] c responses to UTP were unaected by this treatment. 5 ECV304 cells responded to the diadenosine polyphosphate Ap 3 A with rises in [Ca 2+ ] c . Apparent responses to Ap 4 A, Ap 5 A and Ap 6 A, 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 P2Y 2 receptor insensitive to the diadenosine polyphosphates and a P2Y 1 receptor sensitive to Ap 3 A. In addition, ECV304 cells respond to AMP with increases in [Ca 2+ ] c via an as yet uncharacterized receptor.

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.