The potential cytoprotective actions of a novel nicotinic agent 2,4-dimethoxybenzilidene anabaseine (DMXB) were investigated in differentiated PC12 cells and transected rat septal cholinergic neurons in vivo. In NGF-differentiated PC12 cells, removal of both NGF and serum led to cell loss, a reduced % of cells expressing neurites, the release of lactate dehydrogenase, and a decrease in total cellular protein. Cell loss was apparent within 24 h, and remained constant between 4-8 days post-NGF removal. NGF alone (100 ng/ml), DMXB (10 pM), but not nicotine (10 pM), prevented these cell and neurite losses. DMXB-induced cytoprotection was blocked'by 1 pM mecamylamine. DMXB (1 mg/kg, ip) injected twice but not once per day protected cholinesterasestaining septal neurons from retrograde degeneration following unilateral fimbrial transections. The twice per day DMXB injection-protocol also decreased cell roundness among cholinesterasestaining cells in the lesioned septal hemisphere compared to saline-injected animals. These studies suggest that DMXB may exert cytoprotective activity in NGF-sensitive neuronal populations.

Several carbostyril-based beta-agonists have been shown to bind tightly to and slowly dissociate from the beta2-adrenoceptor (beta2AR). In the present study, the structural features of 8-hydroxy-5-[2-[(1-phenyl-2-methylprop-2-yl)amino]-1-hydroxyethyl] -carbostyril (11a) which contribute to its binding properties at the beta2AR were investigated using a series of synthesized analogs. The k(off), estimated by the rate of cAMP decline in DDT1 MF-2 (DDT) cells with a reduced receptor density, Ki and ligand-induced receptor reductions were determined. All of the derivatives stimulated cAMP accumulation in DDT cells in the sub to mid nanomolar range and elicited the same maximal stimulation as (-)isoproterenol. Derivatives of 11a with side chain N-substitutions comprising 2-methylbutyl, phenylethyl and isopropyl had higher k(off)-values and lower affinities as compared to 11a. Increasing the number of methylenes between the side chain tertiary alpha carbon and phenyl from 1 in 11a to 3 or reducing the number to 0 also resulted in derivatives with higher k(off)- and Ki-values. In addition, replacement of the 8-hydroxycarbostyril nucleus of 11a with catechol reduced the affinity of the compound for the beta2AR by 48-fold and increased its k(off). Only those derivatives with the lowest k(off)-values induced a decrease in the receptor density of DDT cell membranes following a preincubation and extensive washing. The data show that the 8-hydroxycarbostyril nucleus in conjunction with substitutions on the tertiary alpha carbon of the side chain and positioning of the phenyl group are important characteristics determining the high affinity and slow dissociation of 11a from the beta2AR.

1 The chemoreactive ligands 5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)-amino)-2-methylpropyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)amino)-2-methylprop-2-yl)amino-1-hydroxyethyl)-carbostyril (HCITC) were synthesized and shown to be potent irreversible antagonist and agonist ligands, respectively, for the b-adrenoceptor in DDT 1 MF-2 (DDT) cells and the rat isolated aorta. 3 In the rat isolated aorta, DCITC (0.1 mM) did not a ect either the phenylephrine-mediated tissue contraction or the acetylcholine-mediated relaxation. DCITC attenuated the maximal (7)-isoprenalinemediated relaxation of a phenylephrine contracted aorta in a concentration-dependent manner and shifted the dose-response curves for (7)-isoprenaline to the right. The DCITC-induced decrease in maximal response was not reversed by extensive tissue washing. By use of the operational model of agonism, the calculated dissociation constant for (7)-isoprenaline ws 286 nM and the estimated receptor reserve for this agonist was 23% at the maximal response. 4 HCITC and (7)-isoprenaline stimulated cyclic AMP accumulation in DDT cells with pD 2 values (negative logarithm to base 10 of EC 50 ) of 7.95 and 7.97, respectively, and both mediated the same maximal stimulation. In the rat isolated aorta, HCITC produced a concentration-dependent relaxation of the tissue with a pD 2 value of 6.62, whereas the pD 2 for (7)-isoprenaline was 7.03. However, HCITC produced a greater maximal relaxation of the tissue than (7)-isoprenaline. The HCITC-mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the bantagonist propranolol was added concurrently. In contrast, once the HCITC-mediated responses were established, the addition of propranolol did not result in any attenuation indicating that HCITC is an irreversible b-agonist.