VIP receptor subtypes in mouse cerebral cortex: evidence for a differnntial localization in astrocytes, microvessels and synaptosomal membranes

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[¹²⁵I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studies in primary cultures, indicates the presence of a single class of high affinity binding sites with a K_d of 3.3 nM and a B_max of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[¹²⁵I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis⁴¹. Secretin, despite its lack of interaction with sites labeled by M-[¹²⁵I]VIP, stimulates glycogenolysis with an EC₅₀ of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a K_d of 41.3 nM and a B_max of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[¹²⁵I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with K_d of 1.4 and 30.3 nM, and B_max of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC₅₀ of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a K_d of 4.9 nM and a B_max of 316 fmol/mg protein, and a low affinity class with a K_d of 42.8 nM and a B_max of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[¹²⁵I]VIP with an EC₅₀ of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[¹²⁵I]VIP exclusively for the neuronal class of binding sites.