H Kurose

Perikaryal and synaptic localization ofα2A-adrenergic receptor-like immunoreactivity

Through molecular cloning, the existence of three distinct subtypes ofα2-adrenergic receptors (α2AR) - A, B and C - has been established and are referred to asα2AAR, α2BAR andα2CAR. Due to limitations in pharmacological tools, it has been difficult to ascribe the role of each subtype to the central functions ofα2AR. In situ hybridization studies have provided valuable information regarding their distribution within brain. However, little is known about their subcellular distribution, and in particular, their pre- versus postsynaptic localization or their relation to noradrenergic neurons in the CNS. We used an antiserum that selectively recognizes the A-subtype ofα2AR to determine: (1) the regional distribution of the receptor within brains of rat and monkey; (2) the subcellular distribution of the receptor in locus coeruleus (LC) of rats and prefrontal cortex of monkeys; and (3) the ultrastructural relation of the receptor to noradrenergic processes in LC. Light microscopic immunocytochemistry revealed prominent immunoreactivity in LC, the brainstem regions modulating the baroreflex, the granule cell layer of the cerebellar cortex, the paraventricular and supraoptic nuclei of the hypothalamus (PVN, SON), the basal ganglia, all thalamic nuclei, the hippocampal formation and throughout cerebral cortical areas. Comparison of results obtained from rat and monkey brains revealed no apparent interspecies-differences in the regional distribution of immunoreactivity. Immunoreactivity occurred as small puncta, less than 1 μm in diameter, that cluster over neuronal perikarya. Besides these puncta, cell bodies, proximal dendrites and fine varicose processes - most likely to be axonal - of the PVN and SON and the hippocampal granule cells also exhibited homogeneously intense distribution of immunoreactivity. Subcellularly,α2AAR-ir in LC and prefrontal cortex were associated with synaptic and non-synaptic plasma membrane of dendrites and perikarya as well as perikaryal membranous organelles. In addition, cortical tissue, but not LC, exhibited prominent immunoreactivity within spine heads. Rat brainstem tissue immunolabeled dually forα2AAR and dopamine β-hydroxylase (DβH, the noradrenaline-synthesizing enzyme) revealed thatα2AAR-li occurs in catecholaminergic terminals but is also prevalent within non-catecholaminergic terminals. Terminals exhibitingα2AAR-li formed symmetric and asymmetric types of synapses onto dendrites with and without DβH-immunoreactivity. These results indicate that: (1) the A-subtype ofα2AR is distributed widely within brain; (2)α2AAR-li reflects the presence of newly synthesizedα2AAR in perikarya as well as those receptors along the plasma membrane of perikarya, dendritic trunks and spines; and (3)α2AAR in LC may operate as heteroreceptors on non-catecholaminergic terminals as well as autoreceptors on noradrenergic terminals.

Similar ligand binding in recombinant human α2C2-adrenoceptors produced in mammalian, insect and yeast cells

Abstract Ligand binding properties were investigated in recombinant human α2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisae yeast cells. The expected 43 kDa α2C2 protein was visualized with immunoblotting using a polyclonal α2C2-receptor antibody. [3H]Rauwolscine binding in cell homogenates or membranes (Bmax 3–11 pmol/mg protein; Kd approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (−)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar Ki values in the different host systems. This indicates that α2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.