Background One of the earliest steps in synaptogenesis at the neuromuscular junction is the aggregation of nicotinic acetylcholine receptors at the postsynaptic membrane. This study presents quantitative analyses of receptor and α-Dystroglycan aggregation in response to agrin and laminin-1, alone or in combination. Results Both laminin and agrin increased overall expression of receptors on the plasma membrane. Following a 24 hour exposure, agrin increased the number of receptor aggregates but did not affect the number of α-Dystroglycan aggregates, while the reverse was true of laminin-1. Laminin also increased receptor concentration within aggregates, while agrin had no such effect. Finally, the spatial distribution of aggregates was indistinguishable from random in the case of laminin, while agrin induced aggregates were closer together than predicted by a random model. Conclusions Agrin and laminin-1 both increase acetylcholine receptor aggregate size after 24 hours, but several lines of evidence indicate that this is achieved via different mechanisms. Agrin and laminin had different effects on the number and density of receptor and α-Dystroglycan aggregates. Moreover the random distribution of laminin induced (as opposed to agrin induced) receptor aggregates suggests that the former may influence aggregate size by simple mass action effects due to increased receptor expression.

Background Dinitrosyl nonheme-iron complexes can appear in cells and tissues overproducing nitric oxide. It is believed that due to their chemical nature these species may be implicated in certain pathophysiological events. We studied the possible role of low molecular mass dinitrosyl iron complexes in the control of noradrenaline release in electrically stimulated rat tail artery. Results A model complex, dinitrosyl-iron-thiosulfate (at 1–10 μM) produced a concentration-dependent enhancement of electrical field stimulated [3H]noradrenaline release (up to 2 fold). At the same time, dinitrosyl-iron-thiosulfate inhibited neurogenic vasoconstriction, consistent with its nitric oxide donor properties. A specific inhibitor of cyclic GMP dependent protein kinase, Rp-8pCPT-cGMPS, partially inhibited the effect of dinitrosyl-iron-thiosulfate on neurogenic vasoconstriction, but not on [3H]noradrenaline release. Another model complex, dinitrosyl-iron-cysteine (at 3 μM) elicited similar responses as dinitrosyl-iron-thiosulfate. Conventional NO and NO+ donors such as sodium nitroprusside, S-nitroso-L-cysteine or S-nitroso-glutathione (at 10 μM) had no effect on [3H]noradrenaline release, though they potently decreased electrically-induced vasoconstriction. The "false complex", iron(II)-thiosulfate showed no activity. Conclusions Low molecular mass iron dinitrosyl complexes can up-regulate the stimulation-evoked release of vascular [3H]noradrenaline, apparently independently of their NO donor properties. This finding may have important implications in inflammatory tissues.

Background The mammalian homologue of Seven in Absentia (Siah) can act in the ubiquitin/proteasome pathway. Recent work has shown that Siah can bind group I metabotropic glutamate receptors (mGluRs), but the functional consequences of this interaction are unknown. Results The effects of coexpression of Siah on group I mGluR signaling were examined using heterologous expression in rat sympathetic, superior cervical ganglion neurons. Siah1a attenuated heterologously expressed group I mGluR-mediated calcium current inhibition, but was without effect on group II mGluR- or NE-mediated calcium current modulation via heterologously expressed mGluR2 or native a2 adrenergic receptors, respectively, indicating that the effect of Siah was specific for group I mGluRs. Surface expression and subcellular distribution of group I mGluRs were not detectably altered in the presence of Siah1a as assessed by immunoflourescence experiments with epitope tagged receptors and imaging of a GFP/mGluR fusion construct. In addition, an N-terminal Siah deletion construct, which cannot function in the proteolysis pathway, displayed effects similar to the wild type Siah1a. Finally, coexpression of calmodulin, which competes with Siah1a for binding to the C-terminal tail of group I mGluRs, reversed the effect of Siah1a on mGluR-mediated signaling. Conclusions These data supported the conclusion that the attenuation of mGluR signaling induced by Siah1a expression was likely a direct consequence of Siah/mGluR association rather than a result of targeting of the receptors to the proteosome. In addition, the data suggest that the binding of CaM and Siah may play an important role in the regulation of group I mGluR function.

Background Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. Results In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. Conclusions The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability.

Background The mechanisms by which alpha-2 adrenergic receptors are down-regulated following chronic exposure to agonist are not well understood. Interestingly, the human alpha-2C receptor does not down-regulate, whereas the opossum alpha-2C receptor does down-regulate. A comparison of the amino acid sequence of the third intracellular loop of these two receptors shows that the opossum alpha-2C receptor contains a potential G protein-coupled receptor kinase (GRK)phosphorylation motif (EESSTSE) with four hydroxyl residues, whereas the human alpha-2C receptor motif only contains two hydroxyl residues (DESSAAAAE). Because a similar acidic serine-rich motif (EESSSSD) in the human alpha-2 adrenergic receptor has been demonstrated to be phosphorylated by GRK and all four serines are required for desensitization of the receptor, we sought to determine whether the EESSTSE sequence was involved in the down-regulation of the alpha-2C adrenergic receptor. Results Site-directed mutagenesis was used to mutate the opossum alpha-2C receptor to SSVA and AAVA in place of the SSTS wild-type sequence. Down-regulation experiments on CHO cells transfected with the receptors demonstrated that neither of the mutated receptors down-regulated following 24 h exposure to norepinephrine, whereas the wild-type receptor down-regulated to 65 ± 10% of the control. Conclusions These results indicate that a motif with four hydroxyl amino acid residues in an acidic environment is important for down-regulation of the opossum alpha-2C adrenergic receptor. Because these are potential GRK phosphorylation sites, we suggest that GRK phosphorylation may be involved in alpha-2C adrenergic receptor down-regulation.

Background Previous work has suggested that in the liver, adenosine preconditioning is mediated by nitric oxide. Whether the endothelial isoform of nitric oxide synthase plays a part in this mechanism has however not yet been investigated. Methods Wistar rats were used (6 in each group) – Groups: (1) sham, (2) ischemia-reperfusion, (3) adenosine + ischemia-reperfusion, (4) endothelial isoform inhibitor + adenosine + ischemia-reperfusion. Results Using immunohistochemistry, this study has revealed a decrease in the expression of endothelial nitric oxide synthase following hepatic ischemia-reperfusion. This was prevented by adenosine pre-treatment. When an inhibitor of endothelial nitric oxide synthase was administered prior to adenosine pre-treatment, pre-conditioning did not occur despite normal expression of endothelial nitric oxide synthase. Conclusions These findings suggest that adenosine attenuates hepatic injury by preventing the downregulation of endothelial nitric oxide synthase that occurs during ischemia-reperfusion.

Background Substance P (SP) is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. Results Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. Conclusions The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling.

Background Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. Results We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis . Conclusions Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.