Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation.

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.

Ionizing radiation stimulates existing signal transduction pathways involving the activation of epidermal growth factor receptor and ERBB-3, and changes of intracellular calcium in A431 human squamous carcinoma cells.

Previous studies demonstrated that ionizing radiation activates the epidermal growth factor receptor (EGFR), as measured by Tyr autophosphorylation, and induces transient increases in cytosolic free [Ca2+], [Ca2+]f. The mechanistic linkage between these events has been investigated in A431 squamous carcinoma cells with the EGFR Tyr kinase inhibitor, AG1478. EGFR autophosphorylation induced by radiation at doses of 0.5-5 Gy or EGF concentrations of 1-10 ng/ml is inhibited by >75% at 100 nM AG1478. Activation of EGFR enhances IP3 production as a result of phospholipase C (PLC) activation. At the doses used, radiation stimulates Tyr phosphorylation of both, PLCgamma and erbB-3, and also mediates the association between erbB-3 and PLCgamma not previously described. The increased erbB-3 Tyr phosphorylation is to a significant extent due to transactivation by EGFR as >70% of radiation- and EGF-induced erbB-3 Tyr phosphorylation is inhibited by AG 1478. The radiation-induced changes in [Ca2+]f are dependent upon EGFR, erbB-3 and PLCgamma activation since radiation stimulated IP3 formation and Ca2+ oscillations are inhibited by AG1478, the PLCgamma inhibitor U73122 or neutralizing antibody against an extracellular epitope of erbB-3. These results demonstrate that radiation induces qualitatively and quantitatively similar responses to EGF in stimulation of the plasma membrane-associated receptor Tyr kinases and immediate downstream effectors, such as PLCgamma and Ca2+.

Evaluation of the cannabinoid CB2 receptor-selective antagonist, SR144528: further evidence for cannabinoid CB2 receptor absence in the rat central nervous system.

The aim of this study was to characterize the activity of the cannabinoid CB2 receptor selective antagonist, N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le-3-carboxamide] (SR144528) in a number of biochemical assays and to look for evidence of cannabinoid CB2 receptors in the rat central nervous system. SR144528 displaced [3H]CP 55,940 ((-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxyprop yl]cyclohexan-1-ol) from binding sites in CB2- and CB1-transfected cells (Ki = 0.67+/-0.30 and 33.0+/-5.09 nM) and from rat cerebellum and whole brain membrane homogenates (Ki = 54.7+/-9.70 and 54.8+/-7.86 nM). In the GTPgammaS binding assay, SR144528 antagonized a number of cannabinoid receptor agonists (K(B) values ranging from 26.3 to 76.6 nM) in rat cerebellar membranes and in rat whole brain membranes (K(B) = 50.8 nM). SR144528 also antagonized CP 55,940-stimulated GTPgammaS binding in a CB2-expressing cell line (K(B) = 6.34 nM). In Xenopus oocytes co-expressing the CB1 receptor and G-protein coupled inwardly rectifying K+ channels (GIRK 1/4), SR144528 antagonized WIN 55212-2((R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone) -stimulated K+ currents (K(B) = 558 nM). In summary, this report characterizes the cannabinoid CB2 receptor-selective cannabinoid antagonist, SR144528, and additionally suggests an absence of cannabinoid CB2 receptors in the rat central nervous system, an observation confirmed by Northern blot.

An investigation into the structural determinants of cannabinoid receptor ligand efficacy.

1. A number of side-chain analogues of delta8-THC were tested in GTPgammaS binding assay in rat cerebellar membranes. O-1125, a saturated side-chain compound stimulated GTPgammaS binding with an Emax of 165.0%, and an EC50 of 17.4 nM. 2. O-1236, O-1237 and O-1238, three-enyl derivatives containing a cis carbon-carbon double bond in the side-chain, stimulated GTPgammaS binding, acting as partial agonists with Emax values ranging from 51.3-87.5% and EC50 values between 4.4 and 29.7 nM. 3. The stimulatory effects of O-1125, O-1236, O-1237 and O-1238 on GTPgammaS binding were antagonized by the CB1 receptor antagonist SR 141716A. The K(B) values obtained ranged from 0.11-0.21 mM, suggesting an action at CB1 receptors. 4. Five-ynyl derivatives (O-584, O-806, O-823, O-1176 and O-1184), each containing a carbon-carbon triple bond in the side-chain, did not stimulate GTPgammaS binding and were tested as potential cannabinoid receptor antagonists. 5. Each -ynyl compound antagonized the stimulatory effects of four cannabinoid receptor agonists on GTPgammaS binding. The K(B) values obtained, all found to be in the nanomolar range, did not differ between agonists or from cerebellar binding affinity. 6. In conclusion, alterations of the side-chain of the classical cannabinoid structure may exert a large influence on affinity and efficacy at the CB1 receptor. 7. Furthermore, this study confirms the ability of the GTPgammaS binding assay to assess discrete differences in ligand efficacies which potentially may not be observed using alternative functional assays, thus providing a unique tool for the assessment of the molecular mechanisms underlying ligand efficacies.

Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation.

Accelerated cellular repopulation has been described as a response of tumors to fractionated irradiation in both normal tissue and tumor systems. To identify the mechanisms by which cells enhance their proliferative rate in response to clinically used doses of ionizing radiation (IR) we have studied human mammary and squamous carcinoma cells which are autocrine growth regulated by the epidermal growth factor receptor (EGFR) and its ligands, transforming growth factor-alpha and EGF. Both EGF and IR induced EGFR autophosphorylation, comparable levels of phospholipase C gamma activation as measured by inositol-1,4,5-triphosphate production, and as a consequence oscillations in cytosolic [Ca2+]. Activities of Raf-1 and mitogen-activated protein kinase (MAPK) were also stimulated by EGF and IR by Ca(2+)-dependent mechanisms. All these responses to EGF and IR were dependent upon activation of EGFR as judged by the use of the specific inhibitor of EGFR autophosphorylation, tyrphostin AG1478. Importantly, IR-induced proliferation of A431 cells was also inhibited by AG1478. This is the first report which demonstrates a link between IR-induced activation of proliferative signal transduction pathways and enhanced proliferation. We propose that accelerated repopulation of tumors whose growth is regulated by EGFR is initiated by an IR-induced EGFR activation mechanism that mimics the effects of growth factors.