Biomedical subjects
Publications and source records attributed to A Witter.
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Distribution within the brain of a behaviorally potent [3H]ACTH4-9 analog 2 h after intraventricular injection in rats was studied in the presence and absence of behaviorally and structurally similar peptides, to explore the significance of earlier found preferential uptake of the [3H]ACTH4-9 analog in the septal area. Hypophysectomy resulted in significantly enhanced uptake of radioactivity in the septum as compared to normal rats. No increase in this brain area of hypophysectomized rats was observed after intraventricular injection of [3H]Phe. Elevated circulating ACTH levels after adrenalectomy seemed too low to compete with the septal uptake of the ACTH4-9 analong. Subcutaneous substitution of hypophysectomized rats with sustained release zinc phosphate preparations of the behaviorally equipotent peptides ACTH1-24 and ACTH4-10 decreased the accumulation of the [3H]ACTH4-9 analog in the septum, whereas treatment with the behaviorally inactive fragment ACTH11-24 is not effective. Retreatment of hypophysectomized rats with neuropeptides, differing structurally from natural ACTH peptides (7-D-Phe-ACTH4-10, BETA-LPH61-76 and 9-desglycinamide, 8-Lys-vasopressin), did not change the uptake of the ACTH4-9 analog in any of the investigated brain areas. These results give evidence for specific uptake of the ACTH4-9 analog in the septal region, because competitive displacement occurs only with peptides which both behaviorally and structurally are closely related to the ACTH4-9 analog.
Distribution within the brain of a 3-fold modified ACTH4-9 analog with a remarkably potentiated behavioral activity, 4-MET (O2), 8-d-Lys, 9-Phe-ACTH4-9, was investigated. The radioactive labeled [7-3H-Phe]ACTH4-9 analog was administered intraventricularly in urethane anesthetized rats in a dose of approximately 170 ng. Total radioactivity in CSF, measured in samples drawn from the cisterna magna, decreased over the period of 0.5-4 h after injection from 51 to 2% of the injected dose. Intraventricular injection of the ACTH4-9 analog resulted in high intact peptide levels in the brain. At 2 h after injection the distribution of radioactivity over 2500 micronm and 300 micronm frontal cut brain slices was rather homogenous. Data from distribution studies over topographically defined gross brain structures indicated that the septal area, which is involved in eliciting behavioral activities of ACTH-like neuropeptides, accumulated most of the injected radioactivity per gram wet weight. The distribution profiles within the brain of the [3H]ACTH4-9 analog and [3H]Phe showed considerable differences. Uptake studies in various brain nuclei after intraventricular administration of the [3H]ACTH4-9 analog demonstrated that the greatest part of the investigated nuclei exhibited relative low or medium uptake of radioactivity. This was also true for hippocampal and thalamic nuclei, which have been suggested as effected sites of action for ACTH peptides. Very high accumulation of radioactivity occurred only in the septal nuclei, particularly the dorsal and fimbrial septal nuclei. The results indicate selective uptake of the ACTH4-9 analog in the septal area, suggesting a possible significance of this area as a site of action of ACTH neuro-peptides.
In vivo fate of a threefold substituted ACTH 4-9 analog with a markedly potentiated behavioral activity, 4-Met(O2), 8-D-Lys, 9-Phe-ACTH 4-9, was investigated. The radioactive labeled [7-3H-Phe] ACTH 4-9 analog was administered IV, SC and orally in a dose of approximately 40 mug. Plasma concentrations of total radioactivity and intact peptide were determined at various periods after administration in urethane anesthetized rats. Oral administration was also performed with conscious animals. Maximal plasma concentrations were found 8 min after SC injection. After oral administration in anesthetized rats maximal plasma levels were reached 8 hr after administration; in conscious animals this took 4 hr. The initial volume of distribution was 5.9% of body weight and the initial half-life (t1/2) for intact peptide 4 min. Shortly after IV and SC administration relatively high and stable plasma levels of intact peptide were obtained, reflecting metabolic stability. This stability was also apparent from the metabolite patterns, which were determined in trichloroacetic acid extracts of plasma and brain by paperchromatography and paperelectrophoresis. The plasma profiles indicated increased stability of the labile 8Lys-9Phe bond by the introduction of an 8D-Lys residue in the peptide analog. Enzymatic attack of the analog took place predominantly at 6His-7Phe and 7Phe-8D-Lys. Formation of tritiated water occcurred in brain and the gastro-intestinal tract and was considerable; proteolysis in these compartments was higher than in plasma. High uptake of radioactivity was found in the kidney, but urinary excretion was low during the first 30 min. Uptake in brain was low and paralleled uptake in cerebrospinal fluid. Intact peptide concentrations/g fresh tissue were in the order of 10(-5)-10(-4) times the administered dose for all three routes.
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The effect of substitutions in adrenocorticotropin (ACTH4-9) on extinction of pole-jumping avoidance behavior in intact rats was investigated systemically at two-dose levels. Simultaneous introduction of 4-methionine sulfoxide and 5-D-lysine, in combination with 9-phenylalanine, led to a 1000-fold increase in behavioral potency. The same substitutions induced a 1000-fold decrease in melanocyte-stimulating hormone activity. Incubations of 14-C-labeled ACTH4-9 analogs, prepared by reductive methylation, were carried out with plasma and brain extracts. The resulting metabolites were separated by paper electrophoresis and paper chromatography. The concentrations of nonmetabolized hexapeptides, which appeared to be almost entirely responsible for behavioral activity, were determined as a function of incubation time. The in vitro half-life of intact hexapeptides correlated with their behavioral activity. Therefore, the increase in behavioral potency as a result of amino acid substitutions can be explained, at least partly, by increased resistance against biotransformation.