A. Witter

Penetration of neurohypophyseal hormones from plasma into cerebrospinal fluid (CSF): Half-times of disappearance of these neuropeptides from CSF

The penetration of neurohypophyseal peptides after peripheral administration into the cerebrospinal fluid (CSF) was studied in freely moving rats. In addition, the clearance of these peptides from CSF was investigated. Increased concentrations of vasopressin (AVP) in CSF were detectable 2 min after s.c. injection of 5.0 micrograms of this peptide. Peak concentration was reached at 5 min after administration and this level declined slowly over the next hour. Administration of 5.0 micrograms oxytocin (OXT) s.c. or i.v. resulted in increased OXT levels in CSF within 10 min after application. After 60 min a significant elevation of OXT in CSF was no longer present. These data reveal that approximately 0.002% of the peripherally applied amount of AVP or OXT reached the central nervous system at 10 min after injection. AVP (2.5 ng) and OXT (5.0 ng) applied into one of the lateral brain ventricles reached the cisternal cavity within 2 min after administration. Both neuropeptides were cleared from the CSF with terminal half-times of 26 and 19 min for AVP and OXT, respectively. The present data demonstrate that neurohypophyseal hormones do cross the blood-brain barrier in amounts obviously sufficient to induce central actions.

Dissociation of the behavioural and endocrine effects of lysine vasopressin by tryptic digestion

1 . Lysine vasopressin induces resistance to extinction of active avoidance behaviour (De Wied, 1971). 2 . Digestion of lysine vasopressin with trypsin almost completely destroys the pressor‐, antidiuretic‐, oxytocic‐ and corticotrophin‐releasing factor activities of lysine vasopressin, but does not materially influence its effect on the maintenance of an avoidance response.

α-Endorphin, γ-endorphin and their Des tyrosine fragments in rat pituitary and brain tissue

Abstract Enkephalins, endorphins and related peptides were determined in pituitary and brain tissue of rats which were killed by decapitation or microwave irradiation. The tissues were heated in 1M acetic acid prior to homogenization and the levels of the various peptides were measured by means of a combination of HPLC and radioimmunoassays. Enkephalin levels in pituitary and brain of irradiation-killed rats were much higher as compared to those in tissue of rats sacrificed by decapitation. Similar data were obtained with respect to pituitary levels of γ-endorphin, des-Tyr-γ-endorphin and des- Tyr-α-endorphin. However, brain levels of α- and γ-endorphin and their respective des-Tyr-fragments were not different with the two methods of sacrifice used. The concentrations of β-endorphin in the pituitary gland were similar in rats killed by microwave irradiation and decapitation, but irradiation showed higher β-endorphin levels in the brain than decapitation. These results suggest that β-endorphin fragments like α- and γ-endorphin and des-Tyr-α- and des-Tyr-γ-endorphin are endogenous peptides in the rat pituitary gland and the brain.

Behavioral profile of ψ-MSH: Relationship with acta and β-endorphin action

Abstract In view of the close structural similarity of the pro-opiocortin fragment γ-MSH and of ACTH/MSH type peptides, the behavioral profile of γ-MSH was explored. Attention was first focussed on behavioral procedures in which ACTH/MSH related neuropeptides have been found effective. Using different procedures to test avoidance behavior, it was found that γ-MSH and ACTH-like neuropeptides had opposite effects. In this respect the activity of γ-MSH resembles that of opiate antagonists rather than that of β-endorphin. Accordingly, ACTH 1–24 -induced excessive grooming which is blocked by opiate antagonists, is attenuated by γ-MSH. In addition, γ-MSH injected into the periaqueductal gray matter of the brainstem of opiate naive rats elicited symptoms reminiscent of those seen after opiate withdrawal. γ-MSH attenuated more or less several effects of intracerebroventricularly administered β-endorphin (e.g. antinociception, hypothermia, α-MSH release) and decreased acquisition of heroin self-administration. Although γ-MSH at rather high doses displaced naloxone from its specific binding sites in brain homogenates, it did not interfere with β-endorphin-induced effects on in vitro muscle preparations (guinea pig ileum, rat rectum). Interestingly, γ-MSH induced relaxation of the rat rectum in vitro . It is postulated that γ-MSH may attenuate β-endorphin-induced effects by acting via γ-MSH receptor sites (functional antagonism), although a pharmacological antagonism cannot be excluded as yet.

Specific uptake of a behaviorally potent [3H]ACTH4–9 analog in the septal area after intraventricular injection in rats

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.

Anterior Pituitary Peptides and Avoidance Acquisition of Hypophysectomized Rats

Publisher Summary The pituitary-adrenal system plays an essential role in the defense mechanism of the organism in response to noxious stimuli. These stimuli, which may be of neurogenic or emotional and of somatic or systemic character, invariably cause the discharge of adrenocorticotropic hormone (ACTH) from the adenohypophysis and the subsequent secretion of adrenocortical hormones. Removal of the whole pituitary gland or the adenohypophysis alone causes a severe deficit in the ability to acquire a conditioned avoidance response. The administration of adrenal maintenance doses of ACTH improves the rate of avoidance acquisition of hypophysectomized or adenohypophysectomized rats. Further studies with the heptapeptide ACTH in hypophysectomized rats revealed that it had no effect on adrenal weight, plasma corticosterone, or thymus weight, indicating the absence of corticotropic activities. Nor does the peptide affect the gonads because atrophy of the testes in the hypophysectomized rat is not influenced by chronic treatment with ACTH. Behavioral deficiency of the hypophysectomized rat certainly is linked in some extent to metabolic derangements and physical weakness that occur as a result of hypophysectomy.

Distribution of a behaviorally highly potent ACTH4-9 analog in rat brain after intraventricular administration.

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.

H-Pro-[3H]Leu-Gly-NH2: Metabolism in Human and Rat Plasma Investigated by High-Pressure Liquid Chromatography

H-Pro-Leu-Gly-NH2 (PLG) was labeled with 3H-leucine by catalytic tritiation of H-Pro-methylallylgylcyl-Gly-NH2 and purified by ion-exchange chromatography. Metabolism of 3H-PLG in rat and human plasma was investigated by reversed phase-paired ion high-pressure liquid chromatography. All possible metabolites could be completely separated within 25 min. Half-lives, based on disappearance of intact 3H-PLG, for in vitro metabolism were 26.4 min (rat) and 5.6 days (human). The only significant metabolite was 3H-leucine. A rate-limiting, species-specific enzyme seems responsible for the initial breakdown of PLG. Disappearance of 3H-PLG from rat plasma, following i.v. administration, proceeded with half-lives of 1.03 min (distribution) and 9.8 min (elimination).

Autoradiographic studies with a behaviorally potent 3H-ACTH4–9 analog in the brain after intraventricular injection in rats

Autoradiographic studies aimed at identifying target cells in the brain for ACTH-like peptides were performed using (3H-7-Phe)-4-Met(O2),8-D-Lys, 9-Phe-ACTH4--9, a behaviorally potent analog of ACTH4--9. The 3H-peptide was injected into the lateral ventricle of hypophysectomized rats that were sacrificed 5, 30, 60, 180, and 240 min later. Dry-mount autoradiograms of brain showed the highest density of silver grains in the ventricular lumen and choroid plexus. In addition, radioactivity penetrated brain tissue as far as 100 microns from the ventricles, and was distributed predominantly over neuropil. Within 5 min after the injection, an intracellular concentration of radioactivity above background levels was observed in a small proportion of cells near the ventricles in the septum, caudate-putamen, preoptic area, hypothalamus, thalamus, amygdala, and hippocampus. The cellular labeling decreased in intensity at greater distances from the injection site and at longer survival intervals, and was no longer evident 4 hr after the injection. The labeled cells were usually small, dark, and often elongated, suggesting that ACTH peptides may act preferentially upon a morphologically distinct class of cells in the brain.

H-Pro-[3H]Leu-Gly-NH2: uptake and metabolism in rat brain.

Abstract: The uptake and metabolism of H‐Pro‐[3H]Leu‐Gly‐NH2 ([3H]PLG) in rat brain was investigated by reverse‐phase paired‐ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal‐cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial‐synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7–7.5. The major labeled metabolite was [3H]leucine, whereas 3H‐labeled Leu‐Gly‐NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2‐terminus and that the dipeptide intermediate H‐Leu‐Gly‐NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.