Heat shock proteins bind calcitonin.

We have demonstrated two heat shock proteins (HSP's) in the human placenta that specifically bind calcitonin. Binding specificity was shown by ligand-affinity chromatography and by competitive binding studies. The HSP's were identified by Western analysis and by amino acid sequence. Our observations are consistent with the hypothesis that HSP's may function by binding and interacting with cellular proteins and peptides during their biogenesis. This interaction may both depend upon and produce conformational changes in these ligands during their intracellular processing. Additionally, HSP-peptide hormone interactions may confound studies designed to investigate classical receptor-hormone interactions.

Long-term effects of leucine-enkephalin on active avoidance responding in mice.

The effects of peripherally administered leucine-enkephalin on the acquisition and retention of active avoidance behavior was studied in mice. Mice received 4 training trials on Day 1, 24 test trials on Day 2, and 10 test trials on Day 5. Leucine-enkephalin impaired the aquisition of avoidance behavior when administered before testing on Day 2. Impairment of retention was demonstrated on Day 2 and Day 5 when leucine-enkephalin was administered immediately after training on Day 1, results indicating the long-term nature of the enkephalin effect. There was no effect of leucine-enkephalin when administered just prior to testing on Day 5. The impairing effect was attenuated but not blocked by naloxone. The pattern of the results from the different tests supports the hypothesis that leucine-enkephalin impairs both acquisition of learning and memory consolidation.

Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism.

Leu-enkephalin (100 micrograms/kg, i.p.) administered to mice 5 min before training in a one way active avoidance task significantly reduced the number of avoidances observed in the peptide treated animals. This impairing action of Leu-enkephalin was partially attenuated by methylnaloxonium (naloxonium), a quarternary form of naloxone with a limited ability to penetrate the blood brain barrier. Passive immunization (i.v.) of mice with a Leu-enkephalin antiserum 4 hrs before training produced an effect on avoidance conditioning that was the opposite to that observed with Leu-enkephalin alone. That is, passive immunization increased the number of avoidances observed in the treated mice. The results suggest that Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism, that leu-enkephalin may have a primary site of action outside the blood brain barrier, and that peripheral Leu-enkephalin systems may normally operate to influence conditioned avoidance behavior.

Central versus peripheral actions of Leu-enkephalin on acquisition of a one-way active avoidance response in rats.

A comparison was carried out of the effectiveness of intracerebroventricular or intraperitoneal administration of Leu-enkephalin to affect acquisition of a one-way active avoidance response, locomotor activity or shock sensitivity. Leu-enkephalin impaired acquisition of the avoidance response only when administered peripherally. In contrast, either central or peripheral administration of Leu-enkephalin increased locomotor activity in the open field, and Leu-enkephalin also produced analgesia when administered directly into the brain. The pattern of results suggests that Leu-enkephalin acts at a peripheral site to influence avoidance conditioning.

Effect of adrenalectomy on the circadian rhythm of LTP.

Long-term potentiation (LTP) of hippocampal granule cells was studied in vivo at various times during the light-dark cycle in both intact control and adrenalectomized (ADX) rats. Both groups of rats demonstrated a circadian rhythm of LTP, however the rhythmicity of the ADX animals was the opposite to that found in intact controls. Thus, in experiments performed during the dark phase, intact control animals demonstrated more LTP than did the ADX animals. During the light period ADX animals demonstrated more LTP than intact controls. These data suggest that adrenal hormones play a role in regulating the circadian rhythm of LTP.

Uptake and binding of [3H]hydrocortisone by various pig brain regions.

The cytosol fraction of septum, hypothalamus, and hippocampus of intact and adrenalectomized pigs possessed greater concentrations of radioactivity than the cytosol fraction of amygdala, pituitary, and cortex after an intraventricular injection of [1,2-3H]hydrocortisone. Nuclear extracts from the same brain regions possessed higher concentrations of radioactivity than nuclear extracts of the other brain regions of intact pigs. Nuclear extracts of amygdala, pituitary and hypothalamus from adrenalectomized pigs exhibited the greatest increase over intact pigs in labeled hormone concentration. When adrenalectomized pigs were administered dexamethasone prior to [3H]hydrocortisone, uptake of label was most depressed in hippocampal cytosol and cuclear extract. Also sensitive to the competitive effects of dexamethasone were septal and pituitary nuclear extracts. In intact pigs, pituitary, hippocampus and cortex exhibited higher ratios of bound to total hormone in the cytososl fraction than the other brain regions. Hippocampal and amygdala cytosol possessed the greater magnitude of increase in the ratio of bound to total hormone in adrenalectomized versus intact pigs. The pituitary, septum, amygdala, and cortex of intact and adrenalectomized pigs possessed a ratio of bound to total hormone in nuclear extract 5-10 times greater than that in hippocampus and hypothalamus. However, the latter two regions exhibited a greater increase in bound: total hormone after administration of labeled hormone to adrenalectomized pigs than nuclear extracts of the other brain regions.

Effects of hippocampal and amygdalar stimulation on uptake and binding of 3H-hydrocortisone in the hypothalamus of the cat.

Stimulation of the hippocampus or amygdala of adrenalectomized cats occurred for 10 sec followed by a 50 sec period of no stimulation, beginning 30 min prior to and ending 30 min after administration of 100 muCi of 3H-hydrocortisone into a lateral ventricle. Sixty min after administration of labeled hormone, the hypothalamus was excised and homogenized. Cytosol and nuclear extract fractions were obtained and analyzed for radioactivity and protein content. Separation of bound from free hydrocortisone was achieved by charcoal and adsorption assay. Results reveal that stimulation of the hippocampus resulted in a greater concentration of 3H-hydrocortisone taken up into hypothalamic cells. Also, a greater percentage of total hormone found in the nuclear extract was assayed as bound 3H-hydrocortisone, and the concentration of bound radioactivity in the nuclear extract was increased over control values. Amygdalar stimulation, in general, yielded results similar to those obtained from control cats. However, although a lesser percentage of total hormone in the hypothalamic cytosol was assayed as bound hormone, there was a greater concentration of nuclear bound hormone than in controls, but less than that determined in the hippocampal stimulation group. These results add to the evidence that hippocampus and amygdala have a modulating influence upon the hypothalamo-hypophyseal axis. They also suggest that one manner in which these limbic structures may influence hypothalamic function is to modulate the uptake and binding of hydrocortisone in hypothalamic cells.

Metyrapone inhibition of 3H-hydrocortisone uptake and binding in various brain regions of the pig.

Metyrapone (MET) inhibition of hydrocortisone (3H-HC) uptake binding in the cytosol and nucleus of various brain regions of the pig was demonstrated in vivo. The hippocampus, hypothalamus and septum were the regions most sensitive to the inhibition by MET. The hippocampus exhibited the greatest reduction in 3H-HC concentration in whole homogenate, bound in cytosol and in nuclei. The anterior pituitary and cerebral cortex were the least sensitive to MET. In vitro incubation of hypothalami with 3H-HC and MET substantiated the results obtained in vivo with regard to the inhibitory action of MET on 3H-HC uptake and binding. These results were interpreted to indicate the MET may act directly in certain brain regions and that this inhibitory aciton has important implications for both experimental and clinical uses of this drug.