The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila.

The Drosophila spineless (ss) gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss(-) mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus. Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved.

Sulfated cholecystokinin (26-33) induces mild taste aversion conditioning in rats when administered by three different routes.

We investigated the ability of sulfated cholecystokinin (26-33) (CCK-8) and cholecystokinin (30-33) (CCK-4) to induce taste aversion or avoidance conditioning (TAC) in a one-bottle testing paradigm after either intravenous (i.v.), intracerebroventricular (i.c.v.), or intraperitoneal (i.p.) administration. Significant TAC was induced by i.p. administration of CCK-8 at 0.1 but not at 0.025, 0.5, or 1.0 micromol/kg; the TAC was not robust and, in this case, not even dose related. I.p. administration of CCK-4 at 0.05, 0.1, 0.5, or 1.0 micromol/kg did not induce TAC, replicating other studies from our lab. Mild but significant TAC was also induced by i.v. administration of CCK-8 (at 0.025 and 1.0 but not 0.1 or 0.5 micromol/kg) but not by i.v. administration of CCK-4 (at 0.05, 0.1, 0.5, or 1.0 micromol/kg). Finally, mild but significant TAC was induced by i.c.v. (i.e., lateral ventricular) administration of CCK-8 (at 0.0015 but not at 0.015 micromol/brain) but not by i.c.v. administration of CCK-4 (at 0.005 or 0.010 micromol/brain). Because CCK-4 failed to induce TAC, CCK-8 apparently induced TAC via all three routes by an action at a CCK(a), not CCK(B), receptor mechanism. Because i.c.v. or i.v. administrations of CCK-8 were not more efficacious than i.p. administration, the taste avoidance induced by i.p. administration of CCK-8 was not so mild simply because it failed to reach a critical central locus adequately or because it failed to be delivered at a critical speed (i.e., via i.v. injections). We demonstrate that CCK-8 can induce mild TAC at either peripheral or central sites and suggest that these effects of CCK-8 may be independent and may be a sign of salience but not necessarily of toxicosis.

Regulation of bHLH-PAS protein subcellular localization during Drosophila embryogenesis.

The Drosophila Single-minded and Tango basic-helix-loop-helix-PAS protein heterodimer controls transcription and embryonic development of the CNS midline cells, while the Trachealess and Tango heterodimer controls tracheal cell and salivary duct transcription and development. Expression of both single-minded and trachealess is highly restricted to their respective cell lineages, however tango is broadly expressed. The developmental control of subcellular localization of these proteins is investigated because of their similarity to the mammalian basic-helix-loop-helix-PAS Aromatic hydrocarbon receptor whose nuclear localization is dependent on ligand binding. Confocal imaging of Single-minded and Trachealess protein localization indicate that they accumulate in cell nuclei when initially synthesized in their respective cell lineages and remain nuclear throughout embryogenesis. Ectopic expression experiments show that Single-minded and Trachealess are localized to nuclei in cells throughout the ectoderm and mesoderm, indicating that nuclear accumulation is not regulated in a cell-specific fashion and unlikely to be ligand dependent. In contrast, nuclear localization of Tango is developmentally regulated; it is localized to the cytoplasm in most cells except the CNS midline, salivary duct, and tracheal cells where it accumulates in nuclei. Genetic and ectopic expression experiments indicate that Tango nuclear localization is dependent on the presence of a basic-helix-loop-helix-PAS protein such as Single-minded or Trachealess. Conversely, Drosophila cell culture experiments show that Single-minded and Trachealess nuclear localization is dependent on Tango since they are cytoplasmic in the absence of Tango. These results suggest a model in which Single-minded and Trachealess dimerize with Tango in the cytoplasm of the CNS midline cells and trachea, respectively, and the dimeric complex accumulates in nuclei in a ligand-independent mode and regulates lineage-specific transcription. The lineage-specific action of Single-minded and Trachealess derives from transcriptional activation of their genes in their respective lineages, not from extracellular signaling.

Several roles of CCKA and CCKB receptor subtypes in CCK-8-induced and LiCl-induced taste aversion conditioning.

Administration of a relatively large IP dose of sulfated cholecystokinin (26-33) (CCK-8; 1.0 mumol/kg) consistently induced moderate taste aversion conditioning (TAC) using a 20-min, one-bottle test in Long-Evans rats. Because CCK-8 has affinity for both CCKA and CCKB receptor subtypes, we wanted to determine the subtype involved in CCK-8-induced TAC. Pretreatment with the selective CCKA antagonist MK-329 (L-364, 718 or devazepide), at doses of 0.1, 1.0, or 10.0 mumol/kg, markedly antagonized (> 70%) CCK-8-induced TAC. Pretreatment with the selective CCKB antagonist L-365,260, at doses of 0.1 or 1.0 mumol/kg, partially antagonized (approximately 50%) CCK-8-induced TAC, although the highest dose of L-365,260. 10.0 mumol/kg, did not. These partial antagonistic effects of L-365,260 on CCK-8-induced TAC were replicated in our second study. In our third study, we observed that another CCKB antagonist, the dipeptoid CI-988, also partially antagonized CCK-8-induced TAC at a dose of 0.1, but not 1.0 or 10.0, mumol/kg. In our final study, pretreatments with a single dose (i.e., 10.0, but not 0.1 or 1.0, mumol/kg) of either MK-329 or L-365,260 were also shown to partially antagonize the formation of moderate TAC induced by treatment with LiCl at 708 mumol/kg. Marked antagonism of LiCl-induced TAC was also observed following pretreatment with the known anxiolytic chlordiazepoxide HCl at 7.4 mumol/kg. Considering the existing data on the induction of TAC by various CCK analogues, we consider an action of CCK-8 on peripheral CCKA, but not CCKB, receptors necessary for the induction of TAC. Our results of partial antagonism of CCK-8- and LiCl-induced TAC by L-365,260, CI-988, or MK-329 suggest, but do not prove, that both CCKA and CCKB mechanisms may be operative during TAC. Because the CCK antagonists affected TAC like chlordiazepoxide, blockade of CCKA and CCKB mechanisms may produce a mild anxiolytic effect.

Modification of receptor selectivity and functional activity in cholecystokinin peptoid ligands.

Hybrid analogs of the cholecystokinin A (CCK-A) receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (1,A-71623) and the CCK-B receptor selective antagonists PD-135118 (2) and CI-988 (3) were prepared. Incorporation of the Lys(Tac) side chain into 2 produced a moderately potent antagonist of CCK-8 in the isolated guinea pig gallbladder (GPGB). Incorporation of the Lys(Tac) side chain into 3 produced the novel agonist analog 7 (EC50 = 28 nM in the GPGB) with excellent affinity for both human CCK-A (IC50 = 12 nM) and CCK-B (IC50 = 17 nM) receptors. Analog 7 was a full agonist (EC50 = 3.5 nM) for calcium mobilization on CHO-K1 cells expressing hCCK-A receptors but a partial agonist on CHO-K1 cells expressing hCCK-B receptors, eliciting a weak agonist response (EC50 = 2800 nM) and antagonizing CCK-8-induced calcium mobilization (KB = 20 nM). Despite this unusual in vitro profile, analog 7 was a potent anorectic agent in rats (ED50 = 30 nmol/kg) following intraperitoneal administration.

The effects of anorectic and aversive agents on deprivation-induced feeding and taste aversion conditioning in rats.

We compared the effects of intraperitoneally administered LiCl (0.5-2830 mumol/kg), sulfated cholecystokinin26-33 (10-1000 nmol/kg; CCK-8), nonsulfated CCK-8 (500 and 1000 nmol/kg), sulfated CCK26-29 (500 and 1000 nmol/kg), CCK30-33 (10-1000 nmol/kg) bombesin (10-1000 nmol/kg; BOM), (dl) fenfluramine HCl (0.9-37.3 mumol/kg; fenfluramine), fluoxetine HCl (2.9-86.7 mumol/kg; fluoxetine), and d-amphetamine sulfate (0.27-10.9 mumol/kg; AMPH) on both 18-hr deprivation-induced feeding and one-bottle, taste aversion conditioning in male, Long-Evans rats. Doses of LiCl > or = 177 mumol/kg (or 7.5 mg/kg) induced significant, dose-related taste aversions, but only doses of LiCl > or = 2123 mumol/kg (90 and 120 mg/kg) induced significant anorexia. CCK-8 induced marked anorexia (at doses > or = 25-50 nmol/kg), but only relatively mild taste aversions which were only statistically significant at the highest dose (1000 nmol/kg). The anorectic effects of CCK-8 at 500 and 1000 nmol/kg, but not at lower doses, lasted at least 3 hr. Sulfated CCK26-29, CCK30-33 and nonsulfated CCK-8 induced neither anorexia nor taste aversion. BOM induced marked anorexia at all doses tested, but did not induce statistically significant taste aversions. The nonpeptidal anorectic compounds, fenfluramine, fluoxetine, and AMPH, induced both dose-related anorexia and taste aversion conditioning. We focus on several issues concerning the interpretation of taste aversion conditioning. Our results challenge any simple relationship between the ability of a compound to induce taste aversion and to decrease feeding.

Multiple, small doses of cholecystokinin octapeptide are more efficacious at inducing taste aversion conditioning than single, large doses.

Using a one-bottle taste aversion conditioning paradigm, sulfated cholecystokinin(26-33) (CCK-8) has again been shown to induce taste aversion conditioning in rats. Even though the effective doses of CCK-8 are relatively high, they do not induce as strong an aversion as has been demonstrated with LiCl. This pharmacodynamic profile of CCK-8 (i.e., relatively moderate, but not strong, taste aversion induction) may result, in part, from its unusual pharmacokinetic profile. CCK-8 seems to have a plasma half-life of just several minutes, whereas LiCl has a plasma half-life of 6 h in rats. In the present study, CCK-8, CCK-4, or LiCl was administered either as single, large doses immediately following consumption of 0.2% sodium saccharin (SACC), or as 10 half-hourly injections of one-tenth the large dose. Presumably, multiple small doses extended the time CCK-8 and CCK-4 were acting in the body, even though the peak plasma concentrations were quantitatively lower than after the large, single doses. Ten injections of CCK-8 of 10 or 100 nmol/kg (11.4 or 114.3 micrograms/kg) induced significantly stronger taste aversions than single injections of the same total dose of 100 or 1000 nmol/kg (114.3 or 1143.3 micrograms/kg), whereas multiple injections of LiCl of 70.8 mumol/kg (3.0 mg/kg x 10) did not induce stronger taste aversions than single injections of 708 mumol/kg (30.0 mg/kg). Neither single nor multiple injections of CCK-4 of 1000 nmol/kg (596.7 micrograms/kg) x 1, or 100 or 1000 nmol/kg (59.7 or 596.7 micrograms/kg) x 10 induced any sign of taste aversion conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)