Cyclic AMP is a universal second messenger, produced by a family of adenylyl cyclase (AC) enzymes. The last three decades have brought a wealth of new information about the regulation of cyclic AMP production by ACs. Nine hormone-sensitive, membrane-bound AC isoforms have been identified in addition to a tenth isoform that lacks membrane spans and more closely resembles the cyanobacterial AC enzymes. New model systems for purifying and characterizing the catalytic domains of AC have led to the crystal structure of these domains and the mapping of numerous interaction sites. However, big hurdles remain in unraveling the roles of individual AC isoforms and their regulation in physiological systems. In this review we explore the latest on AC knockout and overexpression studies to better understand the roles of G protein regulation of ACs in the brain, olfactory bulb, and heart.

The dihydropyridine derivatives CGP 28392 and BAY K 8644 exert a strong positive inotropic effect in mammalian cardiac muscle, presumably by increasing Ca influx during the action potential. Analysis of the drug effects at the level of single Ca channels by means of the patch-clamp method revealed complicated changes in the kinetics of channel gating. The prevailing effect is a prolongation of the mean open time of Ca channels. In addition, the intervals between channel openings can be slightly prolonged. Ensemble averages of single-channel traces showed a concentration-dependent increase in the mean current amplitude by the drugs. In contrast to beta-adrenoceptor agonists, the increase in Ca current by the dihydropyridine derivatives is not associated with an increase in the intracellular cyclic AMP level.

Medial nucleus tractus solitarius (mNTS) neurons express leptin receptors (LepRs), and intra-mNTS delivery of leptin reduces food intake and body weight. Here, the contribution of endogenous LepR signaling in mNTS neurons to energy balance control was examined. Knockdown of LepR in mNTS and area postrema (AP) neurons of rats (LepRKD) via adeno-associated virus short hairpin RNA-interference (AAV-shRNAi) resulted in significant hyperphagia for chow, high-fat, and sucrose diets, yielding increased body weight and adiposity. The chronic hyperphagia of mNTS/AP LepRKD rats is likely mediated by a reduction in leptin potentiation of gastrointestinal satiation signaling, as LepRKD rats showed decreased sensitivity to the intake-reducing effects of cholecystokinin. LepRKD rats showed increased basal AMP-kinase activity in mNTS/AP micropunches, and pharmacological data suggest that this increase provides a likely mechanism for their chronic hyperphagia. Overall these findings demonstrate that LepRs in mNTS and AP neurons are required for normal energy balance control.

Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).

Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the "downstream" lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a "phosphomimetic" (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel "master regulator" of PKA-stimulated adipocyte lipolysis.

The lowest contents of ATP and the lowest ATP/AMP concentration ratios are observed in the molluscan muscles that have very low rates of energy expenditure during contraction. The highest contents of ATP are observed in the extremely aerobic insect flight muscle and the extremely anaerobic pectoral muscle of the pheasant and domestic fowl. In general, the lowest ATP/AMP concentration ratios are observed for muscle in which the variation in the rate of energy utilization is small (e.g. some molluscan muscles, heart muscle); the highest ratios are observed in muscles in which this variation is large (lobster abdominal muscle, pheasant pectoral muscle, some insect flight muscles). This finding is consistent with the proposed role of AMP and the adenylate kinase reaction in the regulation of glycolysis. However, in the flight muscle of the honey-bee the ATP/AMP ratio is very low, so that glycolysis may be regulated by factors other than the variation in AMP concentration. The variation in the contents of arginine phosphate in muscle from the invertebrates is much larger than the variation in creatine phosphate in muscle from the vertebrates. The contents of hexose monophosphates and pyruvate are, in general, higher in the muscles of vertebrates than in those of the invertebrates. The contents of phosphoenolpyruvate are similar in all the muscles investigated, except for the honey-bee in which it is about 4-10-fold higher. The mass-action ratios for the reactions catalysed by phosphoglucoisomerase and adenylate kinase are very similar to the equilibrium constants for these reactions. Further, the variation in the mass-action ratios between muscles is small. It is concluded that these enzymes catalyse reactions close to equilibrium. However, the mass-action ratios for the reactions catalysed by phosphofructokinase and pyruvate kinase are much smaller than the equilibrium constants. The variation in the ratios between different muscles is large. It is concluded that these enzymes catalyse nonequilibrium reactions. Since the variation in the mass-action ratios for the reactions catalysed by the phosphagen kinases (i.e. creatine and arginine phosphokinases) is small, it is suggested that these reactions are close to equilibrium.

The phosphorylation and dephosphorylation of proteins, catalysed by protein kinases and phosphatases, is the major mechanism for the transduction of intracellular signals in eukaryotic organisms. Signalling pathways often comprise multiple phosphorylation/dephosphorylation steps and a long-standing hypothesis to explain this phenomenon is that of the protein kinase cascade, in which a signal is amplified as it is passed from one step in a pathway to the next. This review represents a re-evaluation of this hypothesis, using the signalling network in which the SnRKs [Snf1 (sucrose non-fermenting-1)-related protein kinases] function as an example, but drawing also on the related signalling systems involving Snf1 itself in fungi and AMPK (AMP-activated protein kinase) in animals. In plants, the SnRK family comprises not only SnRK1, but also two other subfamilies, SnRK2 and SnRK3, with a total of 38 members in the model plant Arabidopsis. This may have occurred to enable linking of metabolic and stress signalling. It is concluded that signalling pathways comprise multiple levels not to allow for signal amplification, but to enable linking between pathways to form networks in which key protein kinases, phosphatases and target transcription factors represent hubs on/from which multiple pathways converge and emerge.

Energy-dependent proteolysis in rabbit reticulocyte lysates has an absolute requirement for both ATP and ubiquitin that is believed due to the ATP-dependent covalent conjugation of ubiquitin (Ub) to substrate proteins as a signal event. Recently the enzyme responsible for activation of ubiquitin has been purified to near homogeneity by covalent affinity chromatography. In the present work we demonstrate that the enzyme catalyzes the net reaction (formula: see text); in which both an enzyme-bound COOH-terminal ubiquitin thiolester and a COOH-terminal ubiquitin adenylate are formed. The complex is sufficiently stable to be isolated by exclusion chromatography and contains ubiquitin/AMP in a ratio of 2. The presence of two forms of covalently linked ubiquitin provides a rationale for the observation of both ubiquitin-dependent ATP:PPi and ATP:AMP exchange. Selective derivatization of the thiol site with iodoacetamide results in a modified enzyme capable of catalyzing only ATP:PPi exchange and in forming only ubiquitin adenylate. Derivatization of the thiol site prevents conjugate formation in a reconstituted system, demonstrating that the ubiquitin thiolester is the proximal donor for subsequent conjugate formation. A three-step mechanism is proposed for ubiquitin activation by the enzyme prior to conjugation.

The AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy that appears to have arisen at an early stage during eukaryotic evolution. In 2001 it was shown to be activated by metformin, currently the major drug for treatment for type 2 diabetes. Although the known metabolic effects of AMPK activation are consistent with the idea that it mediates some of the therapeutic benefits of metformin, as discussed below it now appears unlikely that AMPK is the sole target of the drug. AMPK is also activated by several natural plant products derived from traditional medicines, and the mechanisms by which they activate AMPK are discussed. One of these is salicylate, probably the oldest medicinal agent known to humankind. The salicylate prodrug salsalate has been shown to improve metabolic parameters in subjects with insulin resistance and prediabetes, and whether this might be mediated in part by AMPK is discussed. Interestingly, there is evidence that both metformin and aspirin provide some protection against development of cancer in humans, and whether AMPK might be involved in these effects is also discussed.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported from China in January, 2020. SARS-CoV-2 is efficiently transmitted from person to person and, in 2 months, has caused more than 82 000 laboratory-confirmed cases of coronavirus disease 2019 (COVID-19) and 2800 deaths in 46 countries. The total number of cases and deaths has surpassed that of the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV). Although both COVID-19 and severe acute respiratory syndrome (SARS) manifest as pneumonia, COVID-19 is associated with apparently more efficient transmission, fewer cases of diarrhoea, increased mental confusion, and a lower crude fatality rate. However, the underlying virus-host interactive characteristics conferring these observations on transmissibility and clinical manifestations of COVID-19 remain unknown.

Methods: We systematically investigated the cellular susceptibility, species tropism, replication kinetics, and cell damage of SARS-CoV-2 and compared findings with those for SARS-CoV. We compared SARS-CoV-2 and SARS-CoV replication in different cell lines with one-way ANOVA. For the area under the curve comparison between SARS-CoV-2 and SARS-CoV replication in Calu3 (pulmonary) and Caco2 (intestinal) cells, we used Student's t test. We analysed cell damage induced by SARS-CoV-2 and SARS-CoV with one-way ANOVA.

Findings: SARS-CoV-2 infected and replicated to comparable levels in human Caco2 cells and Calu3 cells over a period of 120 h (p=0·52). By contrast, SARS-CoV infected and replicated more efficiently in Caco2 cells than in Calu3 cells under the same multiplicity of infection (p=0·0098). SARS-CoV-2, but not SARS-CoV, replicated modestly in U251 (neuronal) cells (p=0·036). For animal species cell tropism, both SARS-CoV and SARS-CoV-2 replicated in non-human primate, cat, rabbit, and pig cells. SARS-CoV, but not SARS-CoV-2, infected and replicated in Rhinolophus sinicus bat kidney cells. SARS-CoV-2 consistently induced significantly delayed and milder levels of cell damage than did SARS-CoV in non-human primate cells (VeroE6, p=0·016; FRhK4, p=0·0004).

Interpretation: As far as we know, our study presents the first quantitative data for tropism, replication kinetics, and cell damage of SARS-CoV-2. These data provide novel insights into the lower incidence of diarrhoea, decreased disease severity, and reduced mortality in patients with COVID-19, with respect to the pathogenesis and high transmissibility of SARS-CoV-2 compared with SARS-CoV.

Funding: May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Richard Yu and Carol Yu, Michael Seak-Kan Tong, Respiratory Viral Research Foundation, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund, Chan Yin Chuen Memorial Charitable Foundation, Marina Man-Wai Lee, The Hong Kong Hainan Commercial Association South China Microbiology Research Fund, The Jessie & George Ho Charitable Foundation, Perfect Shape Medical, The Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Diseases and Research Capability on Antimicrobial Resistance for the Department of Health of the Hong Kong Special Administrative Region Government, The Theme-Based Research Scheme of the Research Grants Council, Sanming Project of Medicine in Shenzhen, and The High Level-Hospital Program, Health Commission of Guangdong Province, China.

Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.

Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O(2)) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O(2) concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the alpha-subunit of hypoxia-inducible factor 1. At higher O(2) concentrations (1-3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O(2) or O(2)-derived species as signaling molecules. Diversion of O(2) away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells.

In the present report, we show that prostaglandin (PG) E2 is the most potent factor which stimulates aromatase expression via cyclic AMP and promoter II. PGE2 acts via EP1 and EP2 receptor subtypes to stimulate both the PKC and PKA pathways. The combined stimulation of both of these pathways results in maximal expression of promoter II-specific CYP19 transcripts. Since PGE2 is a major secretory product both of breast tumor epithelial cells and fibroblasts, as well as of macrophages infiltrating the tumor site, then this could be the mechanism whereby estrogen biosynthesis is stimulated in breast sites adjacent to a tumor, leading in turn to increased growth and development of the tumor itself.

Acetyl-CoA carboxylase (ACC) is rapidly regulated by reversible phosphorylation; phosphorylation inactivates ACC, whereas dephosphorylation activates the enzyme. Among protein kinases only cAMP-dependent protein kinase and 5'-AMP-dependent protein kinase can inactivate ACC; cAMP-dependent protein kinase phosphorylates Ser-77 and -1200; 5'-AMP-dependent protein kinase phosphorylates Ser-79, -1200, and -1215. In this report, the construction and expression of ACC cDNA containing the entire coding region (7.2 kilobase pairs) is described. In order to identify the critical phosphorylation site(s) for each protein kinase, we introduced site-specific mutations at Ser-77, -79, -1200, and -1215 of ACC cDNA and a series of mutated ACCs containing various combinations of these four mutated sites was expressed. By examination of the various mutant ACCs, we provided evidence that the effect of cAMP-dependent protein kinase is entirely mediated by the phosphorylation of Ser-1200 and that Ser-79 is important for 5'-AMP-dependent protein kinase action in vitro.

Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding (ATF/CREB) family of transcription factors. It is an adaptive-response gene that participates in cellular processes to adapt to extra- and/or intracellular changes, where it transduces signals from various receptors to activate or repress gene expression. Advances made in understanding the immunobiology of Toll-like receptors have recently generated new momentum for the study of ATF3 in immunity. Moreover, the role of ATF3 in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers.

AMP-activated protein kinase (AMPK) plays a key role in maintaining energy homeostasis. Activation of AMPK in peripheral tissues has been shown to alleviate the symptoms of metabolic diseases, such as type 2 diabetes, and consequently AMPK is a target for treatment of these diseases. Recently, a small molecule activator (A-769662) of AMPK was identified that had beneficial effects on metabolism in ob/ob mice. Here we show that A-769662 activates AMPK both allosterically and by inhibiting dephosphorylation of AMPK on Thr-172, similar to the effects of AMP. A-769662 activates AMPK harboring a mutation in the gamma subunit that abolishes activation by AMP. An AMPK complex lacking the glycogen binding domain of the beta subunit abolishes the allosteric effect of A-769662 but not the allosteric activation by AMP. Moreover, mutation of serine 108 to alanine, an autophosphorylation site within the glycogen binding domain of the beta1 subunit, almost completely abolishes activation of AMPK by A-769662 in cells and in vitro, while only partially reducing activation by AMP. Based on our results we propose a model for activation of AMPK by A-769662. Importantly, this model may provide clues for understanding the mechanism by which AMP leads to activation of AMPK, which in turn may help in the identification of other AMPK activators.

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms. Although AMPs have been essentially studied and developed as potential alternatives for fighting infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been regarded as a therapeutic strategy to explore. As human cancer remains a cause of high morbidity and mortality worldwide, an urgent need of new, selective, and more efficient drugs is evident. Even though ACPs are expected to be selective toward tumor cells without impairing the normal body physiological functions, the development of a selective ACP has been a challenge. It is not yet possible to predict antitumor activity based on ACPs structures. ACPs are unique molecules when compared to the actual chemotherapeutic arsenal available for cancer treatment and display a variety of modes of action which in some types of cancer seem to co-exist. Regardless the debate surrounding the definition of structure-activity relationships for ACPs, great effort has been invested in ACP design and the challenge of improving effective killing of tumor cells remains. As detailed studies on ACPs mechanisms of action are crucial for optimizing drug development, in this review we provide an overview of the literature concerning peptides' structure, modes of action, selectivity, and efficacy and also summarize some of the many ACPs studied and/or developed for targeting different solid and hematologic malignancies with special emphasis on the first group. Strategies described for drug development and for increasing peptide selectivity toward specific cells while reducing toxicity are also discussed.

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.

Studies were performed to determine the effects of acute and chronic voluntary periods of exercise on the expression of hippocampal genes. RNAs from rodents exposed to a running wheel for 3, 7 and 28 days were examined using a microarray with 1176 cDNAs expressed primarily in the brain. The expression of selected genes was quantified by Taqman RT-PCR or RNase protection assay. The largest up-regulation was observed in genes involved with synaptic trafficking (synapsin I, synaptotagmin and syntaxin); signal transduction pathways (Ca2+/calmodulin-dependent protein kinase II, CaM-KII; mitogen-activated/extracellular signal-regulated protein kinase, MAP-K/ERK I and II; protein kinase C, PKC-delta) or transcription regulators (cyclic AMP response element binding protein, CREB). Genes associated with the glutamatergic system were up-regulated (N-methyl-d-aspartate receptor, NMDAR-2A and NMDAR-2B and excitatory amino acid carrier 1, EAAC1), while genes related to the gamma-aminobutyric acid (GABA) system were down-regulated (GABAA receptor, glutamate decarboxylase GAD65). Brain-derived neurotrophic factor (BDNF) was the only trophic factor whose gene was consistently up-regulated at all timepoints. These results, together with the fact that most of the genes up-regulated have a recognized interaction with BDNF, suggest a central role for BDNF on the effects of exercise on brain plasticity. The temporal profile of gene expression seems to delineate a mechanism by which specific molecular pathways are activated after exercise performance. For example, the CaM-K signal system seems to be active during acute and chronic periods of exercise, while the MAP-K/ERK system seems more important during long-term exercise.

The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

In sympathetic neurons, catecholamines interact with prejunctional alpha-adrenergic receptors to reduce delivery of transmitter to postjunctional target organs. This autoinhibitory feedback is a general phenomenon seen in diverse neurons containing a variety of transmitters. The underlying mechanisms of alpha-adrenergic inhibition are not clear, although decreases in cyclic AMP and cAMP-mediated phosphorylation have been implicated. We have studied depolarization-induced catecholamine release and calcium-channel currents in frog sympathetic neurons. Here we show that alpha-adrenergic inhibition of transmitter release can be explained by inhibition of Ca2+-channel currents and not by modulation of intracellular proteins. Noradrenaline strongly reduces the activity of N-type Ca2+ channels, the dominant calcium entry pathway triggering sympathetic transmitter release, whereas L-type Ca2+ channels are not significantly inhibited. The down-modulation of N-type channels involves changes in rapid gating kinetics but not in unitary flux. This is the first detailed description of inhibition of a high-voltage activated neuronal Ca2+ channel at the single-channel level. The coupling between alpha-adrenergic receptors and N-type channels involves a G protein, but not a readily diffusible cytoplasmic messenger or protein kinase C, and may be well suited for rapid and spatially localized feedback-control of transmitter release.

West Nile virus (WNV)-mediated neuronal death is a hallmark of WNV meningitis and encephalitis. However, the mechanisms of WNV-induced neuronal damage are not well understood. We investigated WNV neuropathogenesis by using human neuroblastoma cells and primary rat hippocampal neurons. We observed that WNV activates multiple unfolded protein response (UPR) pathways, leading to transcriptional and translational induction of UPR target genes. We evaluated the role of the three major UPR pathways, namely, inositol-requiring enzyme 1-dependent splicing of X box binding protein 1 (XBP1) mRNA, activation of activating transcription factor 6 (ATF6), and protein kinase R-like endoplasmic reticulum (ER) kinase-dependent eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, in WNV-infected cells. We show that XBP1 is nonessential or can be replaced by other UPR pathways in WNV replication. ATF6 was rapidly degraded by proteasomes, consistent with induction of ER stress by WNV. We further observed a transient phosphorylation of eIF2alpha and induction of the proapoptotic cyclic AMP response element-binding transcription factor homologous protein (CHOP). WNV-infected cells exhibited a number of apoptotic phenotypes, such as (i) induction of growth arrest and DNA damage-inducible gene 34, (ii) activation of caspase-3, and (iii) cleavage of poly(ADP-ribose) polymerase. The expression of WNV nonstructural proteins alone was sufficient to induce CHOP expression. Importantly, WNV grew to significantly higher viral titers in chop(-)(/)(-) mouse embryonic fibroblasts (MEFs) than in wild-type MEFs, suggesting that CHOP-dependent premature cell death represents a host defense mechanism to limit viral replication that might also be responsible for the widespread neuronal loss observed in WNV-infected neuronal tissue.