Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography

Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.     

Dexamethasone and corticosterone receptor sites. Differential topographic distribution in rat hippocampus revealed by high resolution autoradiography

High resolution light microscopic autoradiography was used, together with regional surveys and combined acridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of 3H dexamethasone and to compare the topographic pattern of labeling with that of 3H corticosterone. Nuclear uptake of 3H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With 3H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after 3H dexamethasone. With 3H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.     

Nuclear labelling after prolonged H-uridine incorporation as visualized by high resolution autoradiography

Localization of radioactive labelling over the nuclei of BSC₁ cells is visualized after long periods of ³H-5-uridine incubation followed or not followed by periods of postincubation in nonradioactive medium for up to several days, using high resolution autoradiography combined with a preferential staining method for ribonucleoproteins.It is shown that when cells are labelled for 1 or 6 h with ³H-uridine and postincubated with a non-radioactive medium up to several days, there is always some radioactivity present in the nucleolus and nucleoplasm. When sections of cells fixed after 1 h of labelling followed by 24 h of postincubation are treated with RNase, part of the radioactivity found in the nucleus disappears almost completely only after a succeeding DNase digestion.The majority of interchromatin granules are weakly labelled after most incubation times, with the label localized rather at the periphery of clusters of granules, or are unlabelled.The results are discussed in the context of recent biochemical findings. It is proposed that interchromatin granules might represent a structure containing a limited quantity of slowly labelled nuclear RNA.     

Localization of ribosomal RNA genes by high resolution autoradiography

In the myxomycete Physarum polycephalum, the genes for ribosomal RNA replicate to a large extent in the G2 phase of the mitotic cycle. In mitotically synchronous plasmodia this fact allows the rRNA genes to be localized by electron microscopic autoradiography. After labelling with thymidine in the G2 phase, silver grains were concentrated over the fibrillar and not over the granular regions of the nucleoli. This shows the presence of DNA in these fibrillar regions or their immediate vicinity. Following labelling and chase during the G2 period, nucleoli are dispersed during mitosis and silver grains concentrate over some regions of condensed chromatin.     

Regional distribution of enkephalinase in the rat brain by autoradiography

The first visualization of enkephalinase (neutral metalloendopeptidase, E.C.3.4.24.11) in rat brain was obtained by autoradiography, using a new tritiated inhibitor: [3H]N-[( R,S )3-(N-hydroxy) carboxamido-2-benzyl propanoyl]glycine (3H-HCBP-Gly). The preliminary analysis of sections clearly showed a discrete localization of enkephalinase in enkephalin enriched regions, such as caudate nucleus, putamen, globus pallidus, and substantia nigra. Moreover 3H-HCBP-Gly binding also occurred in choroid plexus and spinal cord.     

The kinetics of 35S-labelled cysteine in the hypothalamic-hypophyseal tract of the rat,studied by autoradiography

Summary The fate of ³⁵S-cysteine incorporated into the hypothalamic-hypophyseal neurosecretory system was followed in normal rats as a function of time from 30 min up to 30 hrs after intraperitoneal administration (11 different times, three rats each). Autoradiography and grain counting were employed to determine quantitatively the activities present in: supraoptic nucleus (SON), paraventricular nucleus (PVN), three different sites in the hypothalamic-hypophyseal tract (HHT), corresponding to its initial, middle and late portions, and neurohypophysis (NH). In SON and PVN, the kinetic behaviour of ³⁵S-cysteine was compatible with a two-compartment model, implying decline of tracer activity in accordance with two exponential components. Of these, the slow throughput component had its closely equivalent, apparently independent, counterparts in the HHT and NH. Replicas of the rapidly abating activity peak were seen, superimposed on the slow component, in the HHT with such timing of its appearance at the various sites, as to be consistent with the concept of travel through the HHT, at substantially constant speed and without delay, of material rich in ³⁵S-cysteine that has been primarily synthesized in the SON and PVN (with the transport theory of neurosecretion). This flow was calculated to have a velocity in the order of 0.6 mm per hour. The kinetic phenomena and their association with the supposed sites of synthesis, and transport, of the neurosecretory material are discussed with reference to earlier literature.This work was supported by a grant from the Sigrid Jusélius Foundation, Helsinki     

Potato starch granule nanostructure studied by high resolution non-contact AFM

Surface studies at ambient conditions of potato starch granules subjected to multiple freezing and thawing, performed by a high resolution non-contact atomic force microscopy (nc-AFM), revealed some details of the starch granule nanostructure. After the treatment, a significant separation and a chain-like organisation of the granule surface elements have been observed. An accurate analysis of the granule surface nanostructure with a single amylopectine cluster resolution could be carried out. The oblong nodules of approximately 20-50 nm in diameter have been observed at the surface of the potato starch granules. The same size particles were precipitated by ethanol from gelatinized potato starch suspensions. They were also detected at the surface of oat and wheat starch granules. After multiple freezing and thawing, the eroded potato granule surface revealed a lamellar structure of its interior. The 30-40 nm inter-lamellar distances were estimated by means of nc-AFM. These findings fit previously proposed dimensions of the structural elements in the crystalline region of the starch granule. The observed surface sub-particles might correspond to the single amylopectine side chain clusters bundled into larger blocklets packed in the lamellae within the starch granule. The results supported the blocklet model of the starch granule structure.     

Localization of newly-synthesized DNA in a mammalian cell as visualized by high resolution autoradiography

The localization of newly-replicated DNA in mouse cells of line P815 in culture is studied by high resolution autoradiography. After 20 or 30 sec of incorporation of ${}^3\text{H-thymidine}$, the silver grains are found throughout the nucleus with a relatively higher (about two-fold) density over the peripheral region of the nucleus. After a further 1 h of chase with non-radioactive thymidine, or after 19 h of continuous growth with the radioactive precursor, the pattern of the nuclear distribution of label is not appreciably different from that found after a short pulse. Autoradiography, combined with EDTA staining resulting in a preferential bleaching of the chromatin, reveals after a short pulse of ${}^3\text{H-thymidine}$ that most of the silver grains lie close to, or over, the border zone between condensed chromatin and the interchromatin region. The results are discussed in the context of other recent findings concerning the sites of DNA replication in eukaryotic cells obtained with different cell systems.     

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.     

Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM 125I-[Sar1]-angiotensin II, [3H]-Ultrofilm autoradiography, computerized microdensitometry and comparison with 125I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with 125I-[Sar1]-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.