From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca(2+) concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time-and concentrationdependent fashion without affecting NO synthase (NOS) expression or L-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 M to 1 mM, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 M. Accordingly, the effect of ascorbic acid on Ca 2؉ -dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 M, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.Endothelium-derived nitric oxide (NO) is a potent signaling molecule in the cardiovascular system participating in many processes such as vascular relaxation, inhibition of platelet aggregation, regulation of endothelial cell adhesivity, and preservation of the normal vessel wall structure (1). NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS) 1 that requires Ca 2ϩ /calmodulin, FAD, FMN, and tetrahydrobiopterin as cofactors (2). The endothelial NOS isoform (eNOS) is constitutively expressed and activated upon an increase of intracellular Ca 2ϩ following cell stimulation with agonists such as thrombin and bradykinin or through serine phosphorylation subsequent to cell stimulation with shear stress or insulin (3, 4).Evidence is accumulating that NO determines the antiatherosclerotic properties of the endothelium (5). All major risk factors for atherosclerosis including hypercholesterolemia, hypertension...

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2؉ /calmodulin-dependent protein kinase kinase ␤ (CaMKK␤). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca 2؉ -dependent mechanism involving the thrombin receptor protease-activated receptor 1 and G q -protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKK␤ using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKK␤ was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKK␤ or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca 2؉ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca 2؉ /CaMKK␤ dependent and one AMP/LKB1 dependent.AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits (6, 16). AMPK has been shown to function as a sensor of the energy state of the cell. It is activated by a rise in the AMP/ATP ratio following a fall of intracellular ATP and initiates a series of changes aimed at regulating energy balance at the cellular level. These processes include the inhibition of ATP-requiring anabolic pathways and the stimulation of ATP-generating catabolic pathways as well as changes in gene and protein expression (6, 16). Additionally, AMPK acts as a regulator of the whole-body energy metabolism by mediating the effects of hormones such as adiponectin, leptin, or ghrelin (28).AMPK activation requires phosphorylation of threonine 172 in the activation loop of the ␣ subunit (18). Two AMPKactivating kinases have been identified recently. LKB1, a tumor suppressor kinase, in complex with two accessory subunits, STRAD and MO25, has been shown to phosphorylate AMPK (19,23,38,46). Ca 2ϩ /calmodulin-dependent protein kinase kinase ␤ (CaMKK␤) has also been identified as an AMPK kinase (20,25,45). In addition to phosphorylation, AMPK is allosterically activated by binding of AMP, and this can also promote phosphorylation of threonine 172 (21). However, AMPK can be activated in an AMP-independent manner as shown with hyperosmotic stress or with the antidiabetic drug metformin (14). The finding that CaMKK␤ acts upstream of AMPK suggests that in addition ...

Signaling from collagen and G proteincoupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin ␣ IIb ␤ 3 activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma; (2) by blocking or inhibiting ␣ IIb ␤ 3 ; (3) by blocking P2Y 12 receptors; (4) by suppression of phosphoinositide 3-kinase (PI3K) ␤. In murine blood, absence of PI3K␥ led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3K␤ delayed initial thrombus formation and reduced individual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y 12 or PI3K isoforms, resulting in single platelets that had inactivated ␣ IIb ␤ 3 and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y 12 and both PI3K␤ and PI3K␥ isoforms is required for perpetuated ␣ IIb IntroductionPlatelet plug formation at sites of vascular injury is considered to consist of 3 phases: initiation, propagation, and perpetuation. 1,2 The initiation phase involves platelet adhesion to von Willebrand factor (VWF) and to collagen exposed in the vessel wall. In the propagation phase, activated platelets secrete mediators such as ADP, thromboxane A 2 (TxA 2 ), and Gas6, which activate other platelets to form aggregates. In the perpetuation phase, fibrin formation and less well-understood postaggregation events are assumed to accomplish stabilization of the thrombus.At arterial shear rates, the thrombotic process is initiated by the tethering of platelets via glycoprotein (GP) Ib-IX-V to VWF, bound to collagen. 3 In human and murine platelets, 2 interacting collagen receptors, GPVI and integrin ␣ 2 ␤ 1 , mediate stable adhesion to collagen. [4][5][6][7] The signaling receptor GPVI triggers series of activation events, including integrin ␣ IIb ␤ 3 activation (providing binding sites, for example, for fibrinogen) and Ca 2ϩ mobilization and secretion, which all function to recruit other platelets. 8,9 During the propagation phase of thrombus formation, released ADP and TxA 2 in a paracrine way pursue the activation process, mediated by P2Y 1 , P2Y 12 , and TP␣ receptors. 8,10,11 The Gq-coupled P2Y 1 and TP␣ receptors evoke Ca 2ϩ mobilization and protein kinase C activity. The P2Y 12 signaling pathway involves Gimediated inhibition of adenylyl cyclase, which lowers cyclic AMP and indirectly enhances Ca 2ϩ signal generation. 12-14 P2Y 12 signaling via Gi also leads to activation of the phosphoinositide 3-kinase (PI3K) and protein kinase B signaling pathways. However, it is still unc...

Objective: Adipose tissue-derived factors link non-alcoholic fatty liver disease (NAFLD) with obesity, which has also been reported for circulating chemerin. On the other hand, hepatic chemerin and chemokine-like receptor 1 (CMKLR1) mRNA expression has not yet been studied in an extensively characterized patient collective. Design: This study was cross-sectional and experimental in design. Methods: Liver tissue samples were harvested from 47 subjects and histologically examined according to the NAFLD activity score (NAS). The concentrations of chemerin and CMKLR1 were measured using semi-quantitative real-time PCR, and the concentration of serum chemerin was measured using ELISA. To evaluate potential effects of chemerin and CMKLR1, cultured primary human hepatocytes (PHHs) were exposed to selected metabolites known to play a role in NAFLD (insulin, glucagon, palmitoic acid, and interleukin-6 (IL6)). Results: Chemerin and CMKLR1 mRNA levels were elevated in the human liver. Their expression was correlated with the NAS (R 2 Z0.543; P!0.001 and R 2 Z0.355; PZ0.014 respectively) and was significantly elevated in patients with definite non-alcoholic steatohepatitis (NASH) (P!0.05 respectively). Linear regression analysis confirmed an independent association of liver fibrosis, steatosis, inflammation, and hepatocyte ballooning with hepatic chemerin mRNA expression (P!0.05 respectively). The expression of hepatic chemerin and CMKLR1 was correlated with the measures of obesity (P!0.05). The incubation of PHHs with IL6 significantly increased the expression of CMKLR1 mRNA (PZ0.027), while that of chemerin remained unaffected (PO0.05). None of the other metabolites showed an influence (PO0.05). Conclusion: This is the first study to show that chemerin mRNA expression is significantly elevated in the liver of NASH patients and that CMKLR1 expression is upregulated in liver inflammation, whereby IL6 could play a causal role.