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Localization of mu opioid receptors on the membranes of nerve endings and tanycytes in the guinea-pig median eminence by electron microscopic radioautography.

The high density of opioid-containing nerve endings in the median eminence together with the absence of direct effects of opioids upon pituitary suggest a local action of opioids in the median eminence. The aim of this work was to address the occurrence of mu-opioid binding sites in the median eminence at the electron microscopic level, using the highly selective radioligand [125I]FK 33-824. mu-Opioid receptors were labeled in vitro on slightly prefixed slices of mediobasal hypothalamus. The labeling was essentially detected in the external part of the median eminence. Most of the silver grains overlaid membrane appositions. Two overall types of appositions were concerned: nerve terminal-nerve terminal or nerve terminal-tanycyte. Detailed analysis of the silver grain distribution indicated that mu receptors were observed on membranes of different types of nerve endings but also of tanycytes. All the binding sites were localized out of synaptic junctions since the median eminence is totally devoid of these structures. Our results suggest that in the median eminence, opioid peptides have a paracrine and/or autocrine action occurring at least via mu receptors located on nerve terminals but also on tanycytes.

Animals↗

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography.

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.

Amino Acid Substitution↗

Intercellular contact and cell-surface galactosyl transferase activity (cell culture-mouse-radioautography-contact inhibition-cis-and trans-galactosylation).

Evidence is presented suggesting the presence of galactosyl transferases and galactosyl acceptors on the outer surfaces of intact Balb/c 3T3 cells. In addition, the data indicate that these transferases may only be capable of transferring galactose from uridine diphosphate galactose to galactosyl acceptors on adjacent cells after intercellular contact is made (trans-glycosylation). Intact Balb/c 3T12 cells, by contrast, show no requirement for intercellular contact in order to carry out this reaction suggesting that these cells, which do not exhibit contact inhibition of growth, may be able to transfer galactose to acceptors situated on the same cell as the enzyme (cis-glycosylation). Electrophoretic and radioautographic assays were used to detect surface transferase activities in these two cell lines. Results of experiments on cells from sparse and dense cultures, and under conditions where intercellular contact was regulated, are consistent with the above hypothesis.

Animals↗

Uridine-5-H3 radioautography of the human sex chromatin body.

To obtain an estimate of the rate of RNA synthesis by the heterochromatic sex chromatin body, human female fibroblasts were labeled with uridine-5-H(3) and radioautographed. The number of grains over the sex chromatin body was compared with the number of grains over a comparable area of euchromatin. The ratio was 0.37. When corrected for the higher content of DNA per unit area in heterochromatin, the maximum rate of RNA synthesis by the DNA of the sex chromatin body was approximately 18% of the rate of RNA synthesis by a comparable amount of euchromatin DNA. The rate of RNA synthesis by the sex chromatin body did not increase significantly with partial despiralization of this chromatin at prophase.

Adolescent↗

Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-H3.

Glycogen synthesis was investigated by giving tritium (H(3))-labeled glucose with carrier to fasted rats in vivo or incubating liver slices from fasted rats in vitro using a glucose-H(3)-containing medium. After 15 min or 1 hr, pieces of liver were fixed and radioautographed for light and electron microscopy. In vivo and in vitro, radioautographic reactions appeared over "glycogen areas" and over zones transitional between these areas and ergastoplasm. Treatment of sections by alpha amylase removed all but about 5% of the radioactivity, so that about 95% of it consisted of glycogen (synthesized during the 15 min or 1 hr elapsing after administration of glucose-H(3)). Within glycogen areas and transitional zones, most silver grains were over or very close to glycogen granules and smooth (or partly smooth) vesicles. Presumably, much of the label was added onto growing glycogen granules, in accord with the biochemical view that glycogen may serve as substrate for further glycogen synthesis. The few silver grains located far from glycogen granules-15% at the 15 min interval in vivo-approximated smooth (or partly smooth) vesicles of endoplasmic reticulum. This observation raised the possibility that smooth membranes play a role in glucose uptake at an early stage in de novo formation of glycogen granules.

Amylases↗