Receptors for the serine protease thrombin and for lysophospholipids are coupled to G proteins and control a wide range of cellular functions, including mitogenesis. Activators of these receptors are present in blood, and can enter the brain during central nervous system (CNS) injury. Reactive astrogliosis, a prominent component of CNS injury with potentially harmful consequences, may involve proliferation of astrocytes. In this study, we have examined the expression and activation of protease activated receptors (PARs), lysophosphatidic acid (LPA) receptors, and sphingosine-1-phosphate (S1P) receptors on murine astrocytes. We show that activation of these three receptor classes can lead to astrogliosis in vivo and proliferation of astrocytes in vitro. Cultured murine cortical astrocytes express mRNA for multiple receptor subtypes of PAR (PAR-1-4), LPA (LPA-1-3) and S1P (S1P-1, -3, -4, and -5) receptors.

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca 2؉ . One of these priming steps involves the maintenance of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P 2 ) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P 2 is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P 2 important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P 2 that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4,5-P 2 and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P 2 , showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca 2؉ entry and suggest that plasma membrane PtdIns-4,5-P 2 is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P 2 in some manner alters calcium channel function in chromaffin cells.

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1 ؊/؊ animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMPdependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails.Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, b-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G s -coupled receptors, such as b-adrenergic receptors.

ABSTRACT-Opioid receptor (MOR) agonism induces palatable food consumption principally through modulation of the rewarding properties of food. N-{ [3,5-difluoro-3Ј-(1H-1,2,4-triazol-3-ylis a novel opioid receptor inverse agonist that, on the basis of in vitro affinity assays, is greater than 10-or 50-fold selective for human or rat MOR, respectively, compared with -opioid receptors (KOR) and ␦-opioid receptors (DOR). Likewise, preferential MOR occupancy versus KOR and DOR was observed by autoradiography in brain slices from Long Evans rats dosed orally with the drug. GSK1521498 suppressed nocturnal food consumption of standard or palatable chow in lean and dietinduced obese (DIO) Long Evans rats. Both the dose-response relationship and time course of efficacy in lean rats fed palatable chow correlated with receptor occupancy and the plasma concentration profile of the drug. Chronic oral administration of GSK1521498 induced body weight loss in DIO rats, which comprised fat mass reduction. The reduction in body weight was equivalent to the cumulative reduction in food consumption; thus, the effect of GSK1521498 on body weight is related to inhibition of food consumption. GSK1521498 suppressed the preference for sucrose-containing solutions in lean rats. In operant response models also using lean rats, GSK1521498 reduced the reinforcement efficacy of palatable food reward and enhanced satiety. In conclusion, GSK1521498 is a potent, MOR-selective inverse agonist that modulates the hedonic aspects of ingestion and, therefore, could represent a pharmacological treatment for obesity and binge-eating disorders.