The effects of cholecystokinin octapeptide (CCK-8), and its antagonist proglumide, upon conditioned behavior in the rat was studied. First, the effects of CCK-8 and proglumide upon passive avoidance behavior was investigated. Rats were trained to avoid a darkened chamber by presenting electrical footshock (two seconds of intensity levels) inside the chamber. Directly following the footshock, injections of CCK-8 or proglumide were given, with avoidance behavior measured 24 hours following the injection. CCK-8 was found to produce reductions in the passive avoidance latency at doses ranging from 30 ug/Kg to 500 ug/Kg. This effect was found to be dependent upon the current intensity used during conditioning. The CCK-8 effect was found when the current was at 0.25 mA, but at no other current setting tested. Proglumide (5 mg/Kg) was found to block the CCK-8 effect upon passive avoidance behavior. A lower dose of proglumide (2 mg/Kg) was found to produce reductions in the passive avoidance latency. These results suggest that CCK-8 may play a role in passive avoidance conditioning in rats. The effects of CCK-8 upon an appetitively conditioned behavior were then investigated. Rats were trained to locate and drink from a drinking tube that contained a 10 percent sucrose solution. Following 30 seconds exposure to the solution, injections of CCK-8 were given, with the latency to begin drinking from the tube measured 24 hours later. CCK-8 was found to produce increases in the latency to begin drinking, at doses of 20 ug/Kg and 100 ug/Kg. CCK-8 also produced a reduction in the amount of sucrose solution consumed during the test period. When CCK-8 was given following exposure to regular tap water, no increase in drinking latency or reduction in consumption was found. These results suggest that CCK-8 can act as an aversive stimulus and is capable of producing conditioned taste aversions. The results of this dissertation project demonstrate that CCK-8 can influence the acquisition of conditioned behavior in the rat when the octapeptide is paired with the presentation of an unconditioned stimulus (shock or sucrose).

The synthesis of seventeen novel conformationally constrained analogues of the neurohypophyseal peptide hormone oxytocin is described. Synthesis of the peptides was accomplished using solid-phase synthesis techniques on either Merrifield or p-methyl-benzhydrylamine resin. Cleavage of peptides from the solid support and deprotection were carried out by either ammonolysis followed by treatment with sodium in liquid ammonia or anhydrous HF. Disulfide formation was accomplished by treatment of the deprotected peptide with aqueous potassium ferricyanide. Purification of the peptide analogues involved a combination of either partition and/or size exclusion chromatography followed by reverse-phase high-performance liquid chromatography. Several conformationally constrained unnatural amino acids were incorporated into the synthetic peptides. Two were prepared and incorporated as a mixture of isomers and the resulting peptides were separated and purified by HPLC. The types of analogues prepared fall into three categories: analogues incorporating conformational restrictions in positions 1 and 2; bicyclic oxytocin peptides; oxytocin antagonists with changes at the Asn⁵ residue. The peptides with conformational restrictions at position 1 or 2 are: [Tic²]OT, [DTic²]OT, [DTic²,Thr⁴]OT, [β-MePhe²]OT, [ΔPhe²]OT, [Cys(CH₂)₅¹,Phe²,Thr⁴,Orn⁸]OT and [Pen¹,DPhe²,Thr⁴,Orn⁸]OT. Bicyclic peptide analogues and their monocyclic precursors include: [Mpa¹,Lys⁴,Glu⁵]OT, [Mpa¹,Lys⁴,Glu⁵]OT, [Mpa¹,Glu⁴,Lys⁸]OT, and [Mpa¹,Glu⁴,Lys⁸]OT. Antagonists with changes in the Asn⁵ residue are: [Pen¹,DPhe²,Thr⁴,Thr⁵,Orn⁸]OT; [Pen¹,DPhe²,Thr⁴,Leu⁵,Orn⁸]OT; [Pen¹,DPhe²,Thr⁴,Asp⁵,Orn⁸]OT; and [Pen¹,DPhe²,Thr⁴,Tyr⁵,Orn⁸]OT. Biological assays of these analogues for oxytocic activity in the rat uterus model have shown one of the β-MePhe²-containing peptides, [L-threo-β-MePhe²]OT, to be a very potent agonist and one bicyclic, [Mpa¹,Glu⁴,Lys⁸]OT to be an extremely potent oxytocin antagonist. Initial biophysical investigations employing 250 MHz nuclear magnetic resonance spectroscopy were also undertaken in order to determine possible solution conformations of these peptide analogues.

Marine mussels accumulate amino acids and glucose from seawater against considerable concentration gradients. The principal site for this uptake is the gill. Previous studies using intact, isolated gills from marine mussels have suggested that the transport mechanism involves coupling to Na⁺, similar to the mechanism of secondary active transport of amino acids and glucose in vertebrate epithelia, but until this dissertation there had been no rigorous test of this hypothesis. Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel, Mytilus edulis, by differential and sucrose density centrifugation. The preparation procedure isolated a population of membranes enriched in brush border membrane markers. The transport of amino acids by two pathways, the alanine-lysine pathway (AK) and the alanine-proline pathway (AP), and the uptake of glucose was studied in the BBMV. The mechanism of transport through the three transport pathways is BBMV involved coupling to Na⁺. Concentrative uptake through the AK pathway, which transported alanine and lysine, also occurred in the presence of Li⁺ and K⁺ gradients. This pathway was the major route for alanine transport in BBMV. The AP pathway transported alanine and proline, and was strictly dependent on Na⁺. Glucose transport in gill BBMV resembled quite closely the Na⁺-coupled transport of glucose in vertebrate epithelia in such characteristics as Na⁺ and substrate specifically, and electrogenicity. Transport through the two amino acid uptake pathways (AK and AP) and through the glucose uptake pathway could be described by Michaelis-Menten kinetics, with high substrate affinities (K(t)'s below 10 μM). Furthermore, it is likely that multiple Na⁺ ions are involved in the transport of these amino acids and glucose in mussel gill BBMV. It appears that these transporters are adapted for function at low substrate concentrations and against large concentration gradients.

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