BACKGROUND AND PURPOSEOrexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACHOrexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX1 orexin receptors using KEY RESULTSOrexin stimulation strongly increased PLD activity -even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration-response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCd. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gai/o nor Gbg seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONSStimulation of OX1 receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCd but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCd in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCd. Abbreviations bARK1, b-adrenoceptor kinase 1; c-and nPKC, conventional and novel PKC, respectively; GF109203X (bisindolylmaleimide I, Gö6850), 2-(1-[3-dimethylaminopropyl]-1H-indol-3-yl)-3-(1H-indol-3-yl)-maleimide; GGTI-2133, N-([4-(imidazol-4-yl)methylamino]-2- [1-naphthyl]benzoyl)leucine trifluoroacetate salt; Gö6976, 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo(2,3-a)pyrrolo(3,4-c)carbazole-12-propanenitrile; HBM, HEPES-buffered medium; KAC1-1, a peptide cPKC activator; KIC1-1, a peptide cPKC inhibitor; KAD1-1, a peptide PKCd activator; KAE1-1, a peptide PKCe activator, KIE1-1, a peptide PKCe inhibitor; MAFP, methyl arachidonyl fluorophosphonate; PA, phosphatidic acid; pEC50, -logEC50; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP5K, type I

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Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin-dependent kinase-5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)-induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA-mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post-synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12 h after treatment. Studies with hippocampal neurons in vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and glycol-bis (2-aminoethylether)-N,N,N ',N '-tetra-acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant-negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant-negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA-mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.

It has been proposed that OX 1 orexin receptors and CB 1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX 1 receptors and potentiate OX 1 receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX 1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX 1 -CB 1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX 1 receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB 1 receptor-expressing cells. When OX 1 and CB 1 receptors were expressed in the same cells, OX 1 stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX 1 response as such was fully abolished by specific inhibition of CB 1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest a functional rather than a molecular interaction of OX 1 and CB 1 receptors.