Determination of transmitter function by neuronal activity

The role of neuronal activity in the determination of transmitter function was studied in cultures of dissociated sympathetic neurons from newborn rat superior cervical ganglia. Cholinergic and adrenergic differentiation were assayed by incubating the cultures with radioactive choline and tyrosine and determining the rate of synthesis and accumulation of labelled acetylcholine and catecholamines. As in previous studies, pure neuronal cultures grown in control medium displayed much lower ratios of acetylcholine synthesis to catecholamine synthesis than did sister cultures grown in medium previously conditioned by incubation on appropriate nonneuronal cells (conditioned medium). However, here we report that neurons treated with the depolarizing agents elevated K+ or veratridine, or stimulated directly with electrical current, either before or during application of conditioned medium, displayed up to 300-fold lower acetylcholine/catecholamine ratios than they would have without depolarization, and thus remained primarily adrenergic. Elevated K+ and veratridine produced this effect on cholinergic differentiation without significantly altering neuronal survival. Because depolarization causes Ca2+ entry in a number of cell types, the effects of several Ca2+ agonists and antagonists were investigated. In the presence of the Ca2+ antagonists D600 or Mg2+, K+ did not prevent the induction of cholinergic properties by conditioned medium. Thus depolarization, either steady or accompanying activity, is one of the factors determining whether cultured sympathetic neurons become adrenergic or cholinergic, and this effect may be mediated by Ca2+

On the role of cyclic nucleotides in the transmitter choice made by cultured sympathetic neurons

Previous investigations have established that electrical activity or chronic depolarization influences the development of neonatal rat sympathetic neurons in dissociated cell culture. Depolarization reduces their ability to respond to a cholinergic inducing factor produced by non-neuronal cells, allowing normal adrenergic differentiation to proceed (Walicke, P., R. Campenot, and P. Patterson (1977) Proc. Natl. Acad. Sci. U. S. A. 74: 5767–5771). The present study examines whether the developmental effects of depolarization are mediated through cyclic nucleotides. Addition of dibutyryl cAMP, dibutyryl cGMP, adenosine, prostaglandin E1, and cholera toxin all raise neuronal cyclic nucleotide levels and qualitatively mimic the developmental effects of depolarization. However, the quantitative decrease in acetylcholine production caused by these cyclic nucleotide agents is much smaller than that caused by depolarization. Short (48-hr) exposures to the cyclic nucleotide derivatives do not alter transmitter synthesis, indicating that long term developmental changes are involved. Chronic depolarization with elevated K+ increases neuronal cAMP 2-fold but has little effect on cGMP. The increase in cAMP is maintained during several weeks of depolarization and is present as early as the 3rd day in vitro, preceding the significant alterations in adrenergic and cholinergic differentiation. Exposure to 2 mM theophylline also increases neuronal cAMP, but in contrast to the other agents, it enhances cholinergic differentiation. In combination with elevated Ktheophylline further increases neuronal cAMP but still favors cholinergic differentiation. Thus, although cAMP satisfies some criteria for being the second messenger in the developmental effects of depolarization, several findings are consistent with the nucleotide playing a central role: (i) Depolarization has much larger effects on transmitter choice than the cyclic nucleotide agents and (ii) theophylline can uncouple cyclic nucleotide levels from the developmental events

On the role of cyclic nucleotides in the transmitter choice made by cultured sympathetic neurons

Previous investigations have established that electrical activity or chronic depolarization influences the development of neonatal rat sympathetic neurons in dissociated cell culture. Depolarization reduces their ability to respond to a cholinergic inducing factor produced by non-neuronal cells, allowing normal adrenergic differentiation to proceed (Walicke, P., R. Campenot, and P. Patterson (1977) Proc. Natl. Acad. Sci. U. S. A. 74: 5767–5771). The present study examines whether the developmental effects of depolarization are mediated through cyclic nucleotides. Addition of dibutyryl cAMP, dibutyryl cGMP, adenosine, prostaglandin E1, and cholera toxin all raise neuronal cyclic nucleotide levels and qualitatively mimic the developmental effects of depolarization. However, the quantitative decrease in acetylcholine production caused by these cyclic nucleotide agents is much smaller than that caused by depolarization. Short (48-hr) exposures to the cyclic nucleotide derivatives do not alter transmitter synthesis, indicating that long term developmental changes are involved. Chronic depolarization with elevated K+ increases neuronal cAMP 2-fold but has little effect on cGMP. The increase in cAMP is maintained during several weeks of depolarization and is present as early as the 3rd day in vitro, preceding the significant alterations in adrenergic and cholinergic differentiation. Exposure to 2 mM theophylline also increases neuronal cAMP, but in contrast to the other agents, it enhances cholinergic differentiation. In combination with elevated Ktheophylline further increases neuronal cAMP but still favors cholinergic differentiation. Thus, although cAMP satisfies some criteria for being the second messenger in the developmental effects of depolarization, several findings are consistent with the nucleotide playing a central role: (i) Depolarization has much larger effects on transmitter choice than the cyclic nucleotide agents and (ii) theophylline can uncouple cyclic nucleotide levels from the developmental events

The protective role of sphingosine-1-phosphate against the action of the vascular disrupting agent combretastatin A-4 3-O-phosphate

Solid tumours vary in sensitivity to the vascular disrupting agent combretastatin A-4 3-O-phosphate (CA4P), but underlying factors are poorly understood. The signaling sphingolipid, sphingosine-1-phosphate (S1P), promotes vascular barrier integrity by promoting assembly of VE-cadherin/β-catenin complexes. We tested the hypothesis that tumour pre-treatment with S1P would render tumours less susceptible to CA4P. S1P (1μM) pretreatment attenuated an increase in endothelial cell (HUVEC) monolayer permeability induced by 10μM CA4P. Intravenously administered S1P (8mg/kg/hr for 20 minutes then 2mg/kg/hr for 40 minutes), reduced CA4Pinduced (30mg/kg) blood fow shut-down in fbrosarcoma tumours in SCID mice (n≥7 per group), as measured by tumour retention of an intravenously administered fuorescent lectin. A trend towards in vivo protection was also found using laser Doppler fowmetry. Immunohistochemical staining of tumours ex vivo revealed disrupted patterns of VE-cadherin in vasculature of mice treated with CA4P, which were decreased by pretreatment with S1P. S1P treatment also stabilized N-cadherin junctions between endothelial cells and smooth muscle cells in culture, and stabilized tubulin flaments in HUVEC monolayers. We conclude that the rapid shutdown of tumour microvasculature by CA4P is due in part to disruption of adherens junctions and that S1P has a protective effect on both adherens junctions and the endothelial cell cytoskeleton

Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biologicals or drug delivery investigations

Chondroitin sulfates and their binding molecules in the central nervous system

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases