Muscarinic receptor characteristics and regulation in rat cerebral cortex: changes during development, aging and the oestrous cycle.

The effects of postnatal development, aging and the oestrous cycle on muscarinic acetylcholine receptor (mAChR) properties were examined in in vitro living slices of rat neocortex. Using the hydrophilic antagonist ([3H]NMS) to label cell surface mAChRs, an increase in both Bmax and Kd was found during the first postnatal weeks. These values peaked at between 20-40 days postnatally and then declined to adult levels.

Binding of a biotinylated neurotrophic ACTH(4-9) analogue, Org 2766, to neurofilament-positive cells in primary or cell line cultures.

To study the putative binding sites of the neurotrophic peptide Org 2766, an analogue of ACTH(4-9) [H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH], biotinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4-10 microM of biotinyl-Org 2766 (b-Org 2766). Binding of both N- and C-terminally biotinylated Org 2766 was seen to phase-bright, round cells with thin processes, but not to flat, orthogonal-shaped cells with tapering processes.

Agonist-induced down-regulation of human 5-HT1A and 5-HT2 receptors in Swiss 3T3 cells.

We have used single cell clones of Swiss 3T3 cells transfected with genes for the human 5-HT1A or 5-HT2 receptor to study down-regulation and desensitization. After pre-incubation of the cells with serotonin agonists, a time-dependent decrease in [3H]8-hydroxy-2-(di-n-propylamino) tetralin or [3H]ketanserin binding was observed. The pertussis toxin sensitive, 5-HT mediated inhibition of forskolin-stimulated cAMP accumulation in 5-HT1A receptor transfected cells was diminished by 68% after a 2 h pre-incubation of the cells with 10 microM 5-HT.

Muscarinic receptor regulation and 2nd messenger responses in rat neocortex cultures.

Primary cultures of dissociated cerebral cortex cells were used to characterize the muscarinic acetylcholinergic receptors (mAChR) present and to study receptor down-regulation and receptor mediated 2nd messenger responses induced by muscarinic agonists. Binding of the hydrophilic antagonist [3H]N-methyl scopolamine ([3H]NMS) to the cultured cells was saturated after one hour at 4 degrees C with a Kd of 93 pM and a Bmax of 958 fmol/mg protein. Competition binding studies with several antagonists and agonists indicated that the mAChR present in the culture were of a mixed M1/M3 subtype.

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes.

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction.