A selective polyclonal antibody directed toward the C-terminal decapeptide common to the a subunits of Gq and Gil G proteins (Gaq/Ga,ii,) was prepared and used to investigate the subcellular distribution of these proteins in WRK1 cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified Gaq and Gall proteins and labeled only two bands corresponding to these a subunits.

Among the most conserved regions in the G-proteincoupled receptors is the (N/D)PX 2-3 Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G q ␣ subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G q protein complexes is not sufficient to activate G q and suggest that Asn-391 is specifically involved in G q proteins activation.

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Lung tissue from 18-day-old rat fetuses was cultured in the presence of 1,25-dihydroxyvitamin D₃ – 1,25(OH)₂D₃ –– (10^––9M) and dexamethasone (10^––7M) for 48 h. 1,25(OH)₂D₃ increased the lung content in phospholipids more specifically related to lung surfactant, phosphatidylcholine and phosphatidylglycerol. This increase was similar to that observed with dexamethasone. In addition, unlike dexamethasone, 1,25(OH)₂D₃ stimulated the surfactant release into luminal spaces, as evidenced by light and electron microscopy. Thus, vitamin D₃ might represent an additional factor controlling fetal lung maturation by stimulating phospholipid synthesis and surfactant release from type II cells.

Antidiabetic sulfonylureas act through receptors coupled to ATP-dependent potassium channels. Using the binding of [3Hjglibenclamide, a highly potent sulfonylurea, to rat brain membranes to follow the purification procedure, we extracted from ovine brain, purified, and partially characterized two peptides that are endogenous ligands for the central nervous system sulfonylurea receptors. These peptides, referred to as a and (3 endosulfme, differ by their isoelectric points, the (3 form being more basic. Each form of endosulfine is recognized equally by the sulfonylurea receptors from the central nervous system and from insulin-secreting m3 cells. In the same concentration range that is active on the receptors, 13 endosulfine releases insulin from a 13-cell line. Endosulfine is a good candidate for being implicated in the physiology of 13 cells and their disorders (e.g., type H diabetes) and in certain pathologies related to modifications of ion fluxes.The antidiabetic sulfonylureas (1) are widely used in the management of non-insulin-dependent diabetes mellitus (NIDDM), also referred to as type II diabetes (2). These drugs (3, 4) stimulate insulin secretion from the islets of Langerhans, which in diabetes display a decreased insulin content (5) and an impaired response to glucose (6). The sulfonylureas act at the cell surface of the 13 cell (7) through interaction with specific binding sites (or receptors) present in membranes from insulin-secreting cells or tissues (8-13). Receptors with similar characteristics were also observed in the central nervous system (CNS; refs. 10,14,and 15) and in the myocardial tissue (16). The sulfonylurea receptor is closely associated to ATP-dependent potassium channels (17-19), also involved in glucose-induced insulin secretion (20). The existence of such a receptor led us to suggest that it represents a recognition site for an endogenous ligand (14) that would regulate physiological processes through mechanisms triggered, in a pharmacological context, by the sulfonylureas. Such an approach for the binding sites for morphine led to the discovery of the endorphines (21). In the CNS, the potential endogenous ligand is likely to be present near the receptor. We have shown that such a ligand exists in the rat brain (22). We describe the presence in and the purification from ovine brain of two peptides, representing most likely two forms of the same entity, for which we propose the name "'endosulfine. " Endosulfine is present in the brain of rat and sheep at similar concentrations-i.e., ca 0.1 pmol of glibenclamide equivalent per g of tissue-and in the rat pancreas. Highly purified endosulfine is like sulfonylureas in that, at concentrations where it interacts with the receptors, it is able to induce insulin release from JTC cells in culture.MATERIALS AND METHODS Sulfonylureas. Glibenclamide was provided by the Guidotti laboratory.[3H]Glibenclamide (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50) Ci/mmol; 1 Ci = 37 GBq) was purchased from Hoechst Pharmaceuticals or DuPont.Binding ...