No abstract

In FRTL5 rat thyroid cells, norepinephrine, by interacting with a1-adrenergic receptors, stimulates inositol phosphate formation, through activation of phospholipase C, and arachidonic acid release. Recent studies have shown that GTP-binding proteins couple several types of receptors to phospholipase C activation. The present study was undertaken to determine whether GTP-binding proteins couple a1-adrenergic receptors to stimulation of phospholipase C activity and arachidonic acid release. When introduced into permeabilized show that phospholipase C and phospholipase A2 are activated by a1-adrenergic agonists. Both phospholipases are coupled to the receptor by GTP-binding proteins. That coupled to phospholipase A2 is pertussis toxin-sensitive, whereas that coupled to phospholipase C is pertussis toxin-inseiitive.FRTL5 is a cell line derived from normal rat thyroid in which thyrotropin induces the appearance of a1-adrenergic receptors (1). In these cells a1-adrenergic stimulation evokes enhanced inositol phosphate formation (2) and release of arachidonic acid (3). Inositol phosphates and arachidonic acid released by stimulation of a1-adrenergic receptors are linked to different functions. Inositol phosphates appear to enhance intracellular free calcium to mediate iodine efflux (4), while arachidonic acid via its cyclooxygenase metabolite prostaglandin E2 enhances DNA synthesis (3). Inositol phosphate formation results from phospholipase C activity on phosphatidylinositols. The release of arachidonic acid has been ascribed either to activity of phospholipase A2 (5) or to sequential action of phospholipase C and diacylglycerol lipase (6).The coupling of receptors to phospholipases is unclear. However, recent evidence suggests that GTP-binding proteins [N (or G) proteins] are involved in activation of phospholipases C (7-14). Phospholipases A2 usually are described as having a requirement for calcium, and their activation has been assumed to result from elevation of intracellular calcium induced by receptor agonists. However, the levels of calcium available after receptor activation (high nanomolar to low micromolar) are far below those usually found optimal for phospholipase A2 activation in vitro (several millimolar).The present study was undertaken to gain insight into the mechanism of activation of arachidonic acid release in FRTL5 thyroid cells. We find that activation of N proteins by the GTP analog guanosine 5'-[y-thio]triphosphate (GTP[y-SJ) results in release of arachidonic acid and enhanced inositol phosphate formation. The N proteins mediating these effects are distinct. That mediating arachidonic acid release is pertussis toxin-sensitive, whereas that mediating inositol phosphate release is not affected by pertussis toxin. MATERIALS AND METHODSMaterials. RHC 80267 was a gift of Revlon Health Care Group, Tuckahoe, NY. Norepinephrine, phorbol 12-myristate 13-acetate, 1-oleoyl-2-acetyl-rac-glycerol, neomycin, nifedipine, TMB-8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester], and fatty...

In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E2 (PGE2) synthesis. The EC50 values for stimulation of PGE2 sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate-enhanced bradykinin-stimulated PGE2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykininstimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [3H1choline, the phospholipase A2 products lysophosphatidylcholine and glycerophosphocholine were generated. In cells pretreated with dexamethasone, lysophosphatidylcholine and glycerophosphocholine formation induced by bradykinin were inhibited. Treatment of cells with phorbol ester enhanced bradykinin-induced formation of these metabolites. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A2 and that phospholipase A2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.Recent evidence suggests that GTP-binding proteins (G proteins) are involved in the receptor-mediated activation of phosphatidylinositol-specific phospholipase C (1-5). The identities of the G proteins that couple receptors to phosphatidylinositol-specific phospholipase C are unknown. In some tissues the G proteins that couple receptors to phospholipase C exhibit pertussis toxin sensitivity (1, 2), whereas in other tissues pertussis toxin is without effect (6, 7). In vitro, the GTP-binding proteins G0 and Go appear to activate phospholipase C (5). More recently, it has been recognized that phospholipase A2 is also coupled to receptors by G proteins (8-10). In the systems described thus far, the G proteins coupled to phospholipase A2 have been found to be pertussis toxin-sensitive.Bradykinin stimulates arachidonic acid release and prostaglandin synthesis in a variety of tissues (11). This peptide also increases inositol phosphate (InsP) formation by activating a phosphatidylinositol-specific phospholipase C. It has been suggested that free arachidonate may be released by the action of phosphatidylinositol-specific phospholipase C followed by diglyceride or monoglyceride lipases (12), or that phospholipase A2 is responsible for catalyzing the direct release of arachidonate (13). The mechanism by which bradykinin stimulates arachidonic acid release and metabolism to prostaglandin E2 (PGE2) is unclear.Using Swiss 3T3 cells, the present study was designed to examine whether bradykinin stimulates arachidonate release and PGE2 synthesis through a G protein-mediated mechanism and whether release is catalyzed by a phospholipase C-diglyceride lipase pathway or a phospholipase A2...

In this early POC study, GLYX-13 reduced depressive symptoms within 2 hours and this effect was maintained for 7 days on average in subjects with MDD who had not responded to another antidepressant agent during the current depressive episode. The findings of this study support the hypothesis that modulation of the NMDA receptor is a valid target for the development of antidepressant drugs and the need for additional studies to further evaluate the effects of GLYX-13. POC studies such as the one described here play a pivotal role in allowing drug researchers to decide whether to move forward with larger and more expensive studies, and they enable them to focus available resources on those molecules that appear to have the most therapeutic promise. Based on the POC study described here, a multiple dose study has been completed which showed sustained therapeutic benefit with repeated dosing of GLYX-13 for more than 6 weeks. Phase 3 studies are now being planned.

Introduction The N-methyl-d-aspartate receptor-ionophore complex plays a key role in learning and memory and has efficacy in animals and humans with affective disorders. GLYX-13 is an N-methyl-d-aspartate receptor (NMDAR) glycine-site functional partial agonist and cognitive enhancer that also shows rapid antidepressant activity without psychotomimetic side effects. Areas covered The authors review the mechanism of action of GLYX-13 that was investigated in preclinical studies and evaluated in clinical studies. Specifically, the authors review its pharmacology, pharmacokinetics, and drug safety that were demonstrated in clinical studies. Expert opinion NMDAR full antagonists can produce rapid antidepressant effects in treatment-resistant subjects; however, they are often accompanied by psychotomimetic effects that make chronic use outside of a clinical trial inpatient setting problematic. GLYX-13 appears to exert its antidepressant effects in the frontal cortex via NMDAR-triggered synaptic plasticity. Understanding the mechanistic underpinning of GLYX-13’s antidepressant action should provide both novel insights into the role of the glutamatergic system in depression and identify new targets for therapeutic development.