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The well-known method of sliding-cavity fluid contactors used by Gosting for diffusion measurements and by Tiselius in electrophoresis has found considerable use in low-gravity research. To date, sliding-cavity contactors have been used in liquid diffusion experiments, interfacial transport experiments, biomolecular crystal growth, biphasic extraction, multistage extraction, microencapsulation, seed germination, invertebrate development, and thin-film casting. Sliding-cavity technology has several advantages for spaceflight: it is simple, it accommodates small samples, samples can be fully enclosed, phases can be combined, multiple samples can be processed at high sample density, real-time observations can be made, and mixed and diffused samples can be compared. An analysis of the transport phenomena that govern the sliding-cavity method is offered. During sliding of one liquid over another flow rates between 0.001 and 0.1m/sec are developed, giving Reynolds numbers in the range 0.1-100. Assuming no slip at liquid-solid boundaries shear rates are of the order 1sec(-1). The measured consequence is the transfer of 2-5% of the content of a cavity to the opposite cavity. In the absence of gravity, buoyancy-driven transport is assumed absent. Transport processes are limited to (1) molecular diffusion, in which reactants diffuse toward one another at rates that depend on their diffusion coefficient and concentration gradient (Fick's second law), (2) solutocapillary (Marangoni) flow driven by surface-tension gradients, (3) capillary flow (drop spreading) at liquid-solid three-phase lines leading to immiscible phase demixing, and (4) vapor-phase diffusive mass transfer in evaporative processes. Quantitative treatment of these phenomena has been accomplished over the past few years in low-gravity research in space and on aircraft.

Two-step crystallization experiments were conducted in low gravity employing a liquid-liquid diffusion method in an effort to eliminate problems associated with protein crystal growth under the supersaturating conditions required for nucleation. Experiments were performed in diffusion cells formed by the sliding of blocks on orbit. Step gradient diffusion experiments consisted of first exposing protein solutions in diffusion half-wells for brief periods to initiating buffer solutions of high precipitant concentrations to induce nucleation followed by expoure of the same protein solutions to solutions of lower precepitant concentration to promote growth of induced nuclei into crystals. To avoid convective disturbances that occur when solutions of discrepant densities are interfaced at normal gravity, crystallization of hen egg-white lysozyme and rabbit skeletal muscle aldolase by step gradient diffusion was investigated in low gravity on four NASA space shuttle flights. In general, the largest ctystals of both proteins formed at the highest initiating precipitant concentration used, which is consistent with nuclei formation upon brief exposure to high precipitant concentration, and that these nuclei are competent for sustained growth at lower precipitant concentration. The two-step approach dissociates nucleation events from crystal growth allowing parameters affecting nucleation kinetics such as time, precipitant concentration and temperature of nucleation to be varied separately from conditions used for post-nucleation growth.

Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma x glioma hybrid cell line (NG108-15 cells). The addition of > or = 1 microM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations > or = 500 microM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis of [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP gamma S, 2-methylthio ATP, beta, gamma-imidoATP or 3'-O-(4-benzoyl)benzoylATP, but not CTP, AMP, beta, gamma-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]P(i) or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D.

Extracellular ATP causes an increase in the concentration of cytoplasmic free calcium ([Ca2+]i) in bovine pulmonary-artery endothelial (BPAE) cells that results in the synthesis and release of prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation. We show here that PGI2 release in BPAE cells correlates with the concentration of the fully ionized form of extracellular ATP (ATP4-) and not with the concentration of other ionic forms of ATP. Concentrations as low as 10 nM-ATP4- elicited an increase in PGI2 release [EC50 (concn. giving half-maximal stimulation) 3 microM] in BPAE cells incubated in an iso-osmotic medium, pH 7.4, lacking Ca2+ and Mg2+. When the pH or the Mg2+ concentration of the medium was varied so as to maintain a constant level of ATP4-, while varying the concentration of proton-ATP (HATP3-) or MgATP2- respectively, PGI2 release remained constant. An inhibitory effect of extracellular Mg2+ on PGI2 release could be attributed solely to a decrease in the concentration of ATP4-. In contrast with Mg2+, extracellular Ca2+ stimulated PGI2 release induced by ATP. Several results suggest that extracellular Ca2+ modulates PGI2 release by increasing Ca2+ uptake through an ATP(4-)-activated plasma-membrane channel. In BPAE cells incubated in Ca(2+)-free medium, ATP elicited a transient increase in [Ca2+]i that declined to the basal level within 60 s. In cells incubated in Ca(2+)-containing medium, ATP caused an increase in [Ca2+]i that had two components: a transient peak in [Ca2+]i (0-60 s) and a sustained increase in [Ca2+]i that was maintained for several minutes after ATP addition. Increasing the concentration of extracellular calcium from 0.25 mM to 10 mM had no effect on the transient rise in [Ca2+]i induced by ATP, but significantly enhanced the magnitude of the sustained increase in [Ca2+]i. Alterations in the magnitude of the sustained increase in [Ca2+]i would likely modulate PGI2 release, which was not complete until 2 min after ATP addition. Extracellular Ca2+ also stimulated PGI2 release induced by bradykinin. Bradykinin caused a sustained increase in [Ca2+]i in BPAE cells in the presence of extracellular Ca2+. Finally, the magnitude of PGI2 release induced by UTP, a more potent agonist than ATP, correlated with the concentration of extracellular fully ionized UTP (UTP4-). These findings support the hypothesis that nucleotide receptors in BPAE cells recognize the fully ionized form of ATP and UTP and are coupled to signal-transduction pathways involving the mobilization of intracellular Ca2+, the influx of extracellular Ca2+ and the subsequent release of PGI2.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.

The adrenergic blocking agent methyldopa (Aldomet) is toxic to C-1300 neuroblastoma cells in vivo. Four injections of Aldomet at a dose of 7.5 mg/injection were given over a period of 24 hr to C-1300 neuroblastoma-bearing mice. This treatment killed a significant proportion of the C-1300 neuroblastoma cells. Flow cytometric data suggest that sensitivity of tumor cells to Aldomet is not related to the cell cycle.

Rat C-6 glioma serves as an experimental model for human glioma. C-6 glioma cells carried to high culture passages in medium containing 10% fetal bovine serum when injected in vivo are unresponsive to treatment with the beta-adrenergic agonist isoproterenol and the phosphodiesterase inhibitor papaverine. When C-6 glioma cells are kept in culture in serum-containing medium, beta-adrenergic receptor density falls and, concomitantly, ability to accumulate cyclic adenosine monophosphate in response to stimulation with catecholamines declines. Responsiveness to treatment in vivo with a beta-adrenergic agonist was restored when C-6 glioma cells were cultured in serum-free defined medium prior to systemic injection into rats. Culturing of C-6 glioma cells in serum-free medium significantly increases the number of beta-adrenergic receptors when compared with C-6 glioma cells grown in serum-containing medium.

The sympathetic nervous system modulates the growth of C-1300 mouse neuroblastoma (C-1300 NB) in vivo. We now report that the presence of sympathetic cervical ganglia in cultures containing C-1300 NB or dispersed S-20 neuroblastoma (S-20 NB) cells augments growth of these tumors in vitro. Sympathetic ganglia-conditioned medium increases proliferation and survival of S-20 NB cells, indicating that nervous system-derived growth factors can exert an effect on neoplastic cells.