Intact T cell receptor signaling by CD4(+) T cells cultured in the rotating wall-vessel bioreactor.

T lymphocytes fail to proliferate or secrete cytokines in response to T cell receptor (TCR) agonists during culture in spaceflight or ground-based microgravity analogs such as rotating wall-vessel (RWV) bioreactors. In RWVs, these responses can be rescued by co-stimulation with sub-mitogenic doses of the diacyl glycerol (DAG) mimetic phorbol myristate acetate. Based on this result we hypothesized that TCR activation is abrogated in the RWV due to impaired DAG signaling downstream of the TCR. To test this hypothesis we compared TCR-induced signal transduction by primary, human, CD4(+) T cells in RWV, and static culture. Surprisingly, we found little evidence of impaired DAG signaling in the RWV. Upstream of DAG, the tyrosine phosphorylation of several key components of the TCR-proximal signal was not affected by culture in the RWV. Similarly, the phosphorylation and compartmentalization of ERK and the degradation of IkappaB were unchanged by culture in the RWV indicating that RAS- and PKC-mediated signaling downstream of DAG are also unaffected by simulated microgravity. We conclude from these data that TCR signaling through DAG remains intact during culture in the RWV, and that the loss of functional T cell activation in this venue derives from the affect of simulated microgravity on cellular processes that are independent of the canonical TCR pathway.

Rigidity controls human desmoplastic matrix anisotropy to enable pancreatic cancer cell spread via extracellular signal-regulated kinase 2.

It is predicted that pancreatic ductal adenocarcinoma (PDAC) will become the second most lethal cancer in the US by 2030. PDAC includes a fibrous-like stroma, desmoplasia, encompassing most of the tumor mass, which is produced by cancer-associated fibroblasts (CAFs) and includes their cell-derived extracellular matrices (CDMs). Since elimination of desmoplasia has proven detrimental to patients, CDM reprogramming, as opposed to stromal ablation, is therapeutically desirable. Hence, efforts are being made to harness desmoplasia's anti-tumor functions. We conducted biomechanical manipulations, using variations of pathological and physiological substrates in vitro, to culture patient-harvested CAFs and generate CDMs that restrict PDAC growth and spread. We posited that extrinsic modulation of the environment, via substrate rigidity, influences CAF's cell-intrinsic forces affecting CDM production. Substrates used were polyacrylamide gels of physiological (~1.5 kPa) or pathological (~7 kPa) stiffnesses. Results showed that physiological substrates influenced CAFs to generate CDMs similar to normal/control fibroblasts. We found CDMs to be softer than the corresponding underlying substrates, and CDM fiber anisotropy (i.e., alignment) to be biphasic and informed via substrate-imparted morphological CAF aspect ratios. The biphasic nature of CDM fiber anisotropy was mathematically modeled and proposed a correlation between CAF aspect ratios and CDM alignment; regulated by extrinsic and intrinsic forces to conserve minimal free energy. Biomechanical manipulation of CDMs, generated on physiologically soft substrates, leads to reduction in nuclear translocation of pERK1/2 in KRAS mutated pancreatic cells. ERK2 was found essential for CDM-regulated tumor cell spread. In vitro findings correlated with in vivo observations; nuclear pERK1/2 is significantly high in human PDAC samples. The study suggests that altering underlying substrates enable CAFs to remodel CDMs and restrict pancreatic cancer cell spread in an ERK2 dependent manner.

Dynamic culture in a rotating-wall vessel bioreactor differentially inhibits murine T-lymphocyte activation by mitogenic stimuli upon return to static conditions in a time-dependent manner.

Depressed immune function is a well-documented effect of spaceflight. Both in-flight studies and ground-based studies using microgravity analogs, such as rotating wall vessel (RWV) bioreactors, have demonstrated that mitogen-stimulated T lymphocytes exhibit decreased proliferation, IL-2 secretion, and activation marker expression in true microgravity and the dynamic RWV-culture environment. This study investigates the kinetics of RWV-induced T lymphocyte inhibition by monitoring the ability of Balb/c mouse splenocytes to become activated under static culture conditions after concanavalin A (Con A) stimulation in an RWV. Splenocytes were stimulated with Con A and cultured for up to 24 h in the RWV before being allowed to "recover" under static culture conditions in the continued presence of Con A. The T-lymphocyte fraction of splenocytes was assayed during the recovery period for IL-2 secretion, expansion of the T-lymphocyte population, and expression of the activation marker CD25. Our results indicate that CD25 expression was not affected by any duration of RWV exposure. In contrast, proliferation and IL-2 secretion were inhibited by >8 and 12 h of exposure, respectively. Culture in the RWV for 24 h resulted in a near-complete loss of cellular viability during the recovery period, which was not seen in cells maintained in the RWV for 16 h or less. Taken together, these results indicate that for up to 8 h of RWV culture activation is not significantly impaired upon return to static conditions; longer duration RWV culture results in a gradual loss of activation during the recovery period most likely because of decreased T-cell viability and/or IL-2 production.

Studies on the methodology of the carboxyfluorescein assay and on the mechanism of liposome stabilization by red blood cells in vitro.

The stability of small unilamellar liposomes was investigated in human blood, in vitro. Using the carboxyfluorescein technique, interaction between the dye, the detergent Triton X-100, and an as yet unidentified component of human serum grossly interferes with the experiment and necessitates the use of other detergents, preferably sodium deoxycholate. Separation of liposomes and blood cells by centrifugation induces a small leakage from the liposomes and can lead to an underestimation of the real liposome stability. Upon incubation with whole blood, intact liposomes are adsorbed nonspecifically to erythrocytes and internalized by leukocytes, the extent and kinetics of the former process being insensitive to the presence of metabolic inhibitors. The stability of liposomes is significantly enhanced in whole blood or in serum containing washed erythrocytes. Similarly, liposome stability in serum could be augmented be presaturating the serum lipoproteins with excess phospholipid. Our work adds support to previous notions that stable liposomes with high affinities for certain blood-cell components might be developed as suitable carrier systems for drug targetting in pathological disorders within the blood stream.

Stabilization of large multilamellar liposomes by human serum in vitro.

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3-8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.

Effect of cholesterol on Ca2+-induced aggregation and fusion of sonicated phosphatidylserine/cholesterol vesicles.

Small unilamellar vesicles composed of phosphatidylserine (PS) and cholesterol at various ratios were employed in studying the effect of cholesterol on Ca2+-induced vesicle aggregation and fusion using the Tb/dipicolinic acid assay. The leakage of preencapsulated Tb3+ was also measured. The analysis of the data provided estimates for the rate of aggregation C11, and the rate of fusion per se, f11. An increase in cholesterol contents results in a decrease in C11 values. Similarly, aggregation of PS/cholesterol vesicles is slower than that of PS vesicles in the presence of 650 mM NaCl. With 100 or 200 mM NaCl, the overall fusion rate of PS/cholesterol vesicles is slower than that of PS vesicles; the rate being reduced by an increase in cholesterol contents. With 600 mM NaCl, the overall fusion rate of PS/cholesterol 9:1 vesicles is faster than that of PS vesicles, and results are well-simulated by assuming no delay in vesicle aggregation up to dimers. Emerging f11 values are larger in PS/cholesterol than in PS vesicles. An analysis of fusion kinetics of several lipid concentrations shows that f11 values of PS/cholesterol 3:1 vesicles are 5-times larger than those of PS vesicles, when fusion occurs in a medium containing 200 mM NaCl and 1.5 mM Ca2+. The increase in Na+ concentration from 100 to 200 mM, or 600 mM results in a 50- or 150-fold reduction in f11 values of PS vesicles. It is suggested that incorporation of cholesterol in PS vesicles results in enhancement of Ca2+-induced fusogenic capacity.

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein.

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, gamma-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-alpha-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G(i)-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G(i)-protein.

Synexin-mediated fusion of bovine chromaffin granule ghosts. Effect of pH.

Synexin induces chromaffin granule ghosts to fuse one to another, a process which is followed continuously and quantitatively by monitoring the mixing of the intragranular aqueous compartments. A freeze-thaw technique was used for preparing chromaffin granule ghosts loaded with a self-quenching concentration of the fluorescent, high molecular weight probe FITC-Dextran. When the loaded ghosts were mixed with empty ghosts in the presence of synexin, the two compartments fused, resulting in the dilution of the probe with the concomitant increase in fluorescence. So as to suppress possible leakage signals, anti-fluorescein antibodies which quench probe fluorescence were present in the reaction media. Synexin-mediated fusion of freeze-thaw (F/Th) ghosts and binding of 125I-synexin to these membranes were found to be dependent on Ca2+ concentration, but only in a partial manner. However, these two synexin-mediated properties were demonstrably sensitive to [H+] in the medium. A detailed pH profile of fusion revealed an apparent midpoint of activation at approx. pH 5.2, with asymptotic values at pH 4 (maximum) and pH 7.2 (minimum). In our attempt to determine whether the pH effect was on the synexin or on the membranes, we found that fusion was blocked only by treatment of the membranes with the membrane-impermeant carboxyl group modifier 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. These data suggest that membrane fusion evoked by synexin seems to be promoted by rendering the F/Th membranes relatively less negatively charged while the synexin becomes more positively charged. The fusion process was entirely dependent upon synexin concentration; the k1/2 under optimal conditions of pCa and pH was 85 nM. Similar to what has been previously found with intact granules, an anti-synexin polyclonal antibody partially (48%) blocked fusion, as did pretreatment of the chromaffin granules ghosts with trypsin (30%). We conclude that the coincident pCa and pH sensitivity of synexin-mediated binding to chromaffin granule membranes and their subsequent fusion might be associated with physiological changes in the concentration of both cations in the cytoplasm of secreting chromaffin cells.

Ca2+-independent, protein-mediated fusion of chromaffin granule ghosts with liposomes.

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093-4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca2+, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.

Studies on the mechanism of selective retention of porphyrins and metalloporphyrins by cancer cells.

Comparative studies of the toxicity, stability, and retention of the water-soluble porphyrin, tetraphenylporphyrin sulfonate (TPPS), and its complex with Mn(III), have been made with the human breast cancer cell line MCF-7 wild type, and an adriamycin-resistant line derived from it, termed AdrR. Based on growth inhibition, we determined the maximum non-toxic concentration of MnTPPS tolerated by these cells. The integrity of MnTPPS in vitro was investigated by fluorescence microscopy, and we found that there is very little dissociation of MnTPPS within these cells within 4 days. We report novel proton magnetic resonance relaxation measurements of the bulk water of cells in a gel matrix undergoing perfusion. A slightly greater net uptake of MnTPPS in the wild-type cells was observed compared to AdrR; however, there was no significant difference in retention of MnTPPS. These results indicate that over a period of several hours the mechanism of selective retention of these compounds in tumour cells is not due to specific interaction with heme-binding protein, of which there is enhanced expression in the resistant cells. The fact that the net rate of washout of MnTPPS is approximately the same as the net rate of uptake also appears to eliminate compartmentalization or enzymatic modification of MnTPPS within these cells.

Decreased catecholamine secretion from the adrenal medullae of chronically diabetic BB-Wistar rats.

Many humans with IDDM eventually lose the capacity to secrete epinephrine from their adrenal medullae. The mechanism for this pathological change is unknown. We hypothesized that this abnormality is attributable to neuropathic changes in the greater splanchnic nerves or in the chromaffin cells that they innervate. To study this hypothesis, we isolated rat adrenal glands, perfused them ex vivo, and measured the epinephrine content of the perfusate under various conditions of stimulation. We used transmural electrical stimulation (20-80 V, at 10 Hz) to induce epinephrine secretion indirectly by selectively activating residual splanchnic nerve terminals within the isolated glands. Under these conditions, epinephrine secretion was severely attenuated in glands from female BB-Wistar rats with diabetes of 4 mo duration compared with their age-matched, nondiabetic controls. These perfused diabetic adrenal medullae also demonstrated decreased catecholamine release in response to direct chromaffin cell depolarization with 20 mM K+, evidence that a functional alteration exists within the chromaffin cells themselves. Nonetheless, total catecholamine content of adrenal medullae from these diabetic rats was not significantly different from controls, indicating that the secretory defect was not simply attributable to a difference in the amount of catecholamines stored and available for release. Herein, we also provide histological evidence of degenerative changes within the cholinergic nerve terminals that innervate these glands.

Staphylococcus aureus alpha-toxin activates phospholipases and induces a Ca2+ influx in PC12 cells.

Staphylococcal alpha-toxin at subcytotoxic concentrations stimulated phosphatidylinositol turnover and arachidonic acid release in undifferentiated cultures of pheochromocytoma PC12 cells. Stimulation of phospholipase A2 but not C was dependent on extracellular calcium. Addition of staphylococcal alpha-toxin to PC12 cells caused a dose-dependent, biphasic increase in intracellular calcium measured by fura-2 fluorescence technique. Elevation of intracellular Ca2+ content occurred with a time course similar to those observed for stimulation of phospholipase A2. Alteration of membrane structure and formation of staphylococcal alpha-toxin pores facilitating an influx of Ca2+, represent the probable mechanisms by which phospholipases C and A2 are activated, respectively. These results suggest a possible involvement of Ca2+, phosphoinositides and arachidonic acid metabolites in the pathogenic action of staphylococcus alpha-toxin and caution against the general usage of this toxin as a permeabilizing agent to study stimulus-secretion coupling in secretory cells.

Pardaxin induces aggregation but not fusion of phosphatidylserine vesicles.

The effects on membranes of pardaxin, an amphipathic polypeptide, purified from the gland secretion of the Red Sea Moses sole flatfish Pardachirus marmoratus are dose-dependent and range from formation of voltage-gated, cation-selective pores to lysis. We have now investigated the interactions of pardaxin with small unilamellar liposomes. Light scattering showed that pardaxin (10(-7)-10(-9) M) mediated the aggregation of liposomes composed of phosphatidylserine but not of phosphatidylcholine. Aggregation of phosphatidylserine vesicles was impaired by vesicle depolarization. Furthermore, pardaxin-mediated aggregation between fluorescent-labeled PS vesicles was accompanied by leakage of the vesicle contents, and not by fusogenic process within the aggregates. We suggest that pardaxin is a unique polypeptide, that induces vesicle aggregation and membrane destabilization, but not membrane fusion; the mechanism of the aggregation activity of pardaxin is related to its amphipathic properties.

Destabilization of actin filaments as a requirement for the secretion of catecholamines from permeabilized chromaffin cells.

In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secretion coupling, we have previously demonstrated that the actomyosin system might be involved in the transport of cations across the plasma membrane of bovine adrenal chromaffin cells [(1986) J. Biol. Chem. 261, 5745-5750]. To establish whether actin and myosin might also be involved in later stages of the cellular response, we have examined the possible effects of various actin-specific reagents on the calcium-mediated secretion of catecholamines from digitonin-permeabilized cells. F-Actin-destabilizing agents, such as cytochalasin D or DNase 1, were found to promote Ca2+-stimulated (as well as basal) secretion. By contrast, stabilizers, like phalloidin, produced the opposite effect. It is concluded that stimulus-secretion coupling in chromaffin cells might require the reorganization of actin for modulating both ion transport across the plasma membrane and exocytotic secretion per se.

Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells.

Tetanus toxin, a potent neurotoxin which blocks neurotransmitter release in the CNS, also inhibits Ca2+-induced catecholamine release from digitonin-permeabilized, but not from intact bovine chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified tetanus toxin to bovine adrenal chromaffin granules. Tetanus toxin bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of tetanus toxin to brain synaptosomal membranes. We suggest that the toxin-binding site is a glycoconjugate of the G1b type (a polysialoganglioside or a glycoprotein-proteoglycan) which is localized on the cytoplasmic face of the granule membrane and might directly be involved in exocytotic membrane fusion.

Measurement of cell numbers in microtiter culture plates using the fluorescent dye Hoechst 33258.

A sensitive fluorimetric assay based on DNA binding of the fluorescent dye Hoechst 33258 was developed to quantitate cell numbers in microtiter plates. The cells were fixed and incubated with the dye solution. The bound dye was extracted with ethanol and fluorescence was measured in a spectrofluorimeter. The method is sensitive for as little as a thousand cells and is particularly useful for the normalization of enzyme-linked immunoassay (ELISA) data obtained from the same cells. Another major advantage of this new technique is that in contrast to previously described methods, it requires commonly available, inexpensive equipment.

Endothelial cell-lined skeletal muscle ventricles in circulation.

Skeletal muscle ventricles were constructed from the latissimus dorsi in six dogs by wrapping the muscle around a polypropylene mandrel. Jugular vein endothelial cells were harvested enzymatically and grown in tissue culture. After 3 weeks of vascular delay and 4 weeks of electrical conditioning, five skeletal muscle ventricles were seeded with 5 to 8 x 10(6) autologous endothelial cells by percutaneous injection of a cellular suspension into the lumen of the skeletal muscle ventricle; one skeletal muscle ventricle was injected with culture medium alone as an unseeded control. The autologous endothelial cells were all prelabeled with a lipid-bound cellular marker, PKH-26. After an additional 4 weeks of electrical conditioning, the mandrels were removed and the skeletal muscle ventricles were connected to the descending thoracic aorta and activated to contract during cardiac diastole at a 1:2 ratio with the heart. After 3 hours of continuous pumping, mean diastolic pressure was increased by 35% (58 +/- 7 versus 78 +/- 6 mm Hg, p < 0.05). At this time, the skeletal muscle ventricles were excised for histologic examination. Sections stained with hematoxylin and eosin revealed a continuous cellular layer lining the skeletal muscle ventricle; no cells were present on the lumen of the control skeletal muscle ventricle. All seeded skeletal muscle ventricles exhibited fluorescence as a result of the PKH-26 cellular marker. Immunofluorescent staining with antibodies to von Willebrand factor and ultrastructural analysis with an electron microscope confirmed the endothelial character of these cells lining the lumen of the skeletal muscle ventricles. The ability to create endothelial cell-lined muscular pumping chambers holds important implications for the resolution of thrombotic events in cardiac assist devices as well as toward the clinical application of skeletal muscle ventricles.

Comparison of ICAM-1 and VCAM-1 expression in various human endothelial cell types and smooth muscle cells.

The diversity in the local manifestation of inflammatory vascular lesions might be partially attributable to heterogenous cell adhesion molecule (CAM) expression among endothelial cells (EC) derived from different anatomical locations. We compared basal and tumor necrosis factor-alpha (TNFalpha, 0-100 ng/ml, 0-48 h)-induced intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in cultured human aortic EC (HAEC), vena cava EC (HVCEC), dermal microvascular EC (HMVEC), and vena cava smooth muscle cells (HVCSM), using a fluorescent ELISA and the competitive quantitative RT-PCR. We found marked differences in basal ICAM-1 expression, both at the protein and mRNA levels, such that HAEC>HVCEC approximately equal to HMVEC>>HVCSM. Basal VCAM-1 mRNA levels were significantly lower in HVCEC than in HAEC and HVCSM, while protein levels were indistinguishable. TNFalpha-induced ICAM-1 and VCAM-1 levels in all EC were similar and significantly higher than in HVCSM (2.5- and 5-fold, respectively). Dissimilar levels of basal and TNFalpha-induced CAM expression in vascular cells may explain the varied predisposition of different blood vessels to developing certain vasculopathies.

Expression of VACM-1 protein in cultured rat adrenal endothelial cells is linked to the cell cycle.

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a unique arginine vasopressin (AVP) receptor which shares sequence homology with the cullins, genes involved in the regulation of cell cycle transitions. Unlike either cullins or AVP receptors, however, VACM-1 is expressed exclusively in the vascular endothelial cells and in the renal collecting tubule cells. In order to test the hypothesis that the expression of VACM-1 might be correlated with the cell cycle, and to establish an endothelial cell model for the VACM-1 receptor, we examined VACM-1 expression in rat adrenal medulla endothelial cells (RAMEC). Northern and Western blot analyses of mRNA and protein from RAMEC identified presence of 6.4 kb mRNA and a Mr 81 kDa protein, respectively. Immunostaining of RAMEC with anti-VACM-1 antibodies and Western blot analyses indicated that in RAMEC, VACM-1 protein expression is dependent on the cell cycle. VACM-1 protein virtually disappears during the S phase and localizes to the cytosol during cell division and to the cell membrane at the completion of cytokinesis. Furthermore, pretreatment of RAMEC with anti-VACM-1 specific antibodies increased basal levels of Ca2+and attenuated the AVP-dependent increase in cytosolic Ca2+. In summary, these results indicate that VACM-1 protein expression in RAMEC membrane is linked to the cell cycle, and consequently, VACM-1 may be involved in the regulation of cell division.