Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.     

Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells

Agrin released from motor nerve terminals directs differentiation of the vertebrate neuromuscular junction (NMJ). Activity of nitric oxide synthase (NOS), guanylate cyclase (GC), and cyclic GMP-dependent protein kinase (PKG) contributes to agrin signaling in embryonic frog and chick muscle cells. Stimulation of the NO/cyclic GMP (cGMP) pathway in embryos potentiates agrin's ability to aggregate acetylcholine receptors (AChRs) at NMJs. Here we investigated the timing and mechanism of NO and cGMP action. Agrin increased NO levels in mouse C2C12 myotubes. NO donors potentiated agrin-induced AChR aggregation during the first 20 min of agrin treatment, but overnight treatment with NO donors inhibited agrin activity. Adenoviruses encoding siRNAs against each of three NOS isoforms reduced agrin activity, indicating that these isoforms all contribute to agrin signaling. Inhibitors of NOS, GC, or PKG reduced agrin-induced AChR aggregation in mouse muscle cells by ∼ 50%. However, increased activation of the GTPase Rac1, an early step in agrin signaling, was dependent on NOS activity and was mimicked by NO donors and a cGMP analog. Our results indicate that stimulation of the NO/cGMP pathway is important during the first few minutes of agrin signaling and is required for agrin-induced Rac1 activation, a key step leading to reorganization of the actin cytoskeleton and subsequent aggregation of AChRs on the surface of skeletal muscle cells.     

The Drosophila LEM-domain protein MAN1 antagonizes BMP signaling at the neuromuscular junction and the wing crossveins

BMP signaling responses are refined by distinct secreted and intracellular antagonists in different cellular and temporal contexts. Here, we show that the nuclear LEM-domain protein MAN1 is a tissue-specific antagonist of BMP signaling in Drosophila. MAN1 contains two potential Mad-binding sites. We generated MAN1^ΔC mutants, harbouring a MAN1 protein that lacks part of the C-terminus including the RNA recognition motif, a putative Mad-binding domain. MAN1^ΔC mutants show wing crossvein (CV) patterning defects but no detectable alterations in nuclear morphology. MAN1^ΔC pupal wings display expanded phospho-Mad (pMad) accumulation and ectopic expression of the BMP-responsive gene crossveinless-2 (cv-2) indicating that MAN1 restricts BMP signaling. Conversely, MAN1 overexpression in wing imaginal discs inhibited crossvein development and BMP signaling responses. MAN1 is expressed at high levels in pupal wing veins and can be activated in intervein regions by ectopic BMP signaling. The specific upregulation of MAN1 in pupal wing veins may thus represent a negative feedback circuit that limits BMP signaling during CV formation. MAN1^ΔC flies also show reduced locomotor activity, and electrophysiology recordings in MAN1^ΔC larvae uncover a new presynaptic role of MAN1 at the neuromuscular junction (NMJ). Genetic interaction experiments suggest that MAN1 is a BMP signaling antagonist both at the NMJ and during CV formation.     

Ringo/cyclin-dependent kinase and mitogen-activated protein kinase signaling pathways regulate the activity of the cell fate determinant Musashi to promote cell cycle re-entry in Xenopus oocytes

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation.     

Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro.Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [³²P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and ³²P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 M of a synthetic peptide corresponding to the sequence around Ser¹⁰²⁹ of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser¹⁰²⁹ of the STaR/GC remains a candidate phosphorylation site by PKC.Abbreviations STa the heat-stable enterotoxin of E. coli, which has also been called ST-I and STp. The 18 amino acid variant was used throughout - PBS phosphate-buffered saline - PDB 4--12, 13-phorbol dibutyrate - ANP atrial natriuretic peptide - STaR/GC STa receptor/guanylyl cyclase, also called GC-C - PKC protein kinase C     

Specific and nonspecific effects of protein kinase C on the epithelial Na (+) channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na(+) channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49-65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (tau) and a magnitude (DeltaI (V)). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na(+) channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35-45). Stimulation of PKC with 100 nM PMA decreased DeltaI(V) in hENaC-expressing oocytes to a plateau at 57.1 +/- 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (g(Na)) to 21.7 +/- 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g (Na) was attributed to a decrease of membrane capacitance (C (m)), as well as the specific conductance (g(m)/C(m )). The effects on g(m)/C(m) reached a plateau within 15 min, at approximately 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of C(m) was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C(m) were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of g(m ) and by changes of g(m)/C(m), indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.     

Immunohistochemical localization of cyclic AMP and ultrastructural demonstration of adenylate cyclase activity in the testis of Esox lucius at time of spermiation

Summary In the testis of Esox lucius at the time of spermiation, activity of cyclic adenosine 3,5-monophosphate (cAMP) was immunocytochemically localized at the level of the Sertoli cells. In these cells adenylate cyclase activity was also ultracytochemically demonstrated by using adenylyl imidodiphosphate as a substrate. Reaction products of adenylate cyclase were primarily detectable on the basal and adluminal plasma membranes and on the surface of protrusions of the cell body into the lumen.     

Molecular cloning and expression of the catalytic subunit of protein kinase A from Trypanosoma cruzi

The activation of protein kinase A (cyclic adenosine monophosphate-dependent protein kinase) by cyclic adenosine monophosphate is believed to play an important role in regulating the growth and differentiation of Trypanosoma cruzi. A PCR using degenerate oligonucleotide primers against conserved motifs in the VIb and VIII subdomains of the ACG family of serine/threonine protein kinases was utilised to amplify regions corresponding to the parasite homologue of the protein kinase A catalytic subunit. This putative protein kinase A fragment was used to isolate the entire gene from T. cruzi genomic libraries. The deduced 329 amino acid sequence of this gene contained all of the signature motifs of known protein kinase A catalytic subunit proteins. The recombinant protein expressed in Escherichia coli was shown to phosphorylate Kemptide, a synthetic peptide substrate of protein kinase A, in a protein kinase inhibitor (PKI)-inhibitory manner. Immunoprecipitation with polyclonal antisera raised against recombinant protein of this gene was able to pull-down PKI-inhibitory phosphotransferase activity from epimastigote lysates. Immunoblot and Northern blot analyses, in combination with enzyme activity assays, revealed that this gene was a stage-regulated enzyme in T. cruzi with higher levels and activity being present in epimastigotes compared with amastigotes or trypomastigotes. Overall these studies indicate that the cloned gene encodes an authentic protein kinase A catalytic subunit from T. cruzi and are the first demonstration of PKI-inhibitory phosphotransferase activity in an expressed protozoan protein kinase A catalytic subunit.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

Nitric oxide signaling: classical, less classical, and nonclassical mechanisms

Although nitric oxide (NO) was identified more than 150 years ago and its effects were clinically tested in the form of nitroglycerine, it was not until the decades of 1970-1990 that it was described as a gaseous signal transducer. Since then, a canonical pathway linked to cyclic GMP (cGMP) as its quintessential effector has been established, but other modes of action have emerged and are now part of the common body of knowledge within the field. Classical (or canonical) signaling involves the selective activation of soluble guanylate cyclase, the generation of cGMP, and the activation of specific kinases (cGMP-dependent protein kinases) by this cyclic nucleotide. Nonclassical signaling alludes to the formation of NO-induced posttranslational modifications (PTMs), especially S-nitrosylation, S-glutathionylation, and tyrosine nitration. These PTMs are governed by specific biochemical mechanisms as well as by enzymatic systems. In addition, a less classical but equally important pathway is related to the interaction between NO and mitochondrial cytochrome c oxidase, which might have important implications for cell respiration and intermediary metabolism. Cross talk trespassing these necessarily artificial conceptual boundaries is progressively being identified and hence an integrated systems biology approach to the comprehension of NO function will probably emerge in the near future.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.