Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.     

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat

Summary To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2–60 min) after intravenous injection of mono-radioiodinated ¹²⁵I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp⁶, Pro⁹ (Net), DesGly¹⁰]-GnRH produced comparable results. In contrast, the injection of ¹²⁵I-[D-pGlu¹, D-Phe², Trp^3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.     

Internalization of atrial natriuretic factor by AtT-20 corticotropin-secreting cells

Because extended exposure of AtT-20 corticotropin-secreting cells to atrial natriuretic factor (ANF) results in a desensitization of ANF-induced cGMP synthesis, we sought to establish whether pretreatment of AtT-20 cells with the atrial peptide also led to an internalization process. In fact, by coupling an ultrastructural approach to cryoultramicrotomy, ANF-immunoreactivity was detected at both the plasma membrane level and at intracellular sites in AtT-20 cells. Internalization was observed within 5 min at which time labelling was observed in the plasma membrane level, in vacuole-like structures in close proximity to the plasma membrane, in cytoplasmic matrix and sometimes in mitochondria. After 30 min exposure Golgi apparatus, mitochondria and nuclear euchromatin were also labelled. Following 1-4 hr, labelling in other cell compartments, e.g. lysosomal, was increased, while it was reduced in plasma membranes and vacuole-like structures. Secretory granules and endoplasmic reticulum were not labelled throughout the time course. Extraction of a intracellular [125I] ANF from AtT-20 cells following 4 hr incubation suggested that about 90% of the peptide was intact. The data suggest that internalization of ANF may serve to terminate the biological response associated with ANF receptor activation; subcellular distribution of internalized, intact ANF suggests that the peptide may have other, as yet unidentified, intracellular actions.     

A quantitative electron microscope autoradiographic study on 125I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 degrees C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 degrees or 38 degrees C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 degrees than at 38 degrees C. Co-incubation of 125I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with 125I-hCG. The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 degrees C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 degrees C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of 125I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.     

A quantitative electron microscope autoradiographic study on I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 °C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 ° or 38 °C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 ° than at 38 °C. Co-incubation of ¹²⁵I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with ¹²⁵I-hCG.The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 °C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 °C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of ¹²⁵I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.     

An electron-microscope autoradiographic study of transferrin endocytosis by immature erythroid cells

The receptor-mediated endocytosis of 125I-transferrin by immature erythroid cells was studied using the technique of quantitative electron microscope autoradiography. Morphometric analysis of the grain distribution in erythroid cells from the foetal rat liver revealed that the 125I-transferrin radioactivity was localized mainly to intracellular vesicles (61%) and the cell membrane (25%) after 20 min incubation at 37 degrees C. No activity was found associated with the nucleus or mitochondria and only a small amount with the cytosol (13%). In erythroid cells which possessed a prominent Golgi complex, most of the autoradiographic grains were associated with vesicles located in this region, giving rise to a polar distribution of the 125I-transferrin. Uptake of transferrin was found to be maximal at the basophilic normoblast stage of development and then declined progressively during maturation to the reticulocyte. The kinetics of endocytosis of 125I-transferrin by rabbit reticulocytes was also studied by electron microscope autoradiography. Up to 30% of the cell-bound transferrin was internalized almost immediately upon incubation at 37 degrees C. After 30 sec incubation, 42% of the cell-bound 125I-transferrin was estimated to be internal and this rose to almost 70% at steady state between the binding and release of transferrin after 12 min incubation.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

Microbial polysaccharide, HS-142-1, competitively and selectively inhibits ANP binding to its guanylyl cyclase-containing receptor.

During the search for ANP receptor ligands of microbial origin, we isolated a novel polysaccharide, HS-142-1, from culture broth of Aureobasidium sp. HS-142-1 inhibited [125I]-rANP binding to ANP receptor in rabbit kidney cortex membranes with an IC50 of 0.3 mu g/ml, but gave no effects on specific binding of [125I]-Endothelin nor [125I]-Angiotensin II to their respective receptors in bovine lung membranes. HS-142-1 competitively and selectively inhibited ANP binding to its guanylyl cyclase-containing receptor purified from solubilized bovine adrenocortical membranes and blocked cGMP production elicited by ANP. HS-142-1 is the first non-peptide antagonist selective for ANP functional receptor and will be a powerful tool to elucidate the physiological functions of ANP.     

Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.     

Kinetics of internalization and subcellular binding sites for T3 in mouse liver

The intracellular fate of radiolabeled T₃ taken up by mice hepatocytes in vivo was determined at specific time intervals (2–120 min) after injection by quantitative electron microscopic radioautography. Injection of a 200-fold excess of unlabeled T₃ together with [¹²⁵I]-T₃ resulted in a more than 90% inhibition of radioactivity detected in hepatocytes. A simple grain density (GD) analysis of radioautograms revealed that a specific labeling (GD > 1) was displayed by only five cell compartments: the plasma membrane, lipid droplets, mitochondria, nuclear envelope and nuclear matrix whereas other compartments were not labeled. Labeled compartments showed distinct changes in the pattern of labeling over time: the plasma membrane was labeled only 2 min after T₃ injection, whereas labeling of the nuclear envelope was high at 2 min, decreased at 15 min and progressively increased to maximal measured levels at 120 min. After a lag time of 30 min, nuclear matrix labeling increased progressively with time. Mitochondrial labeling was found to be specific at any time point studied but showed no change over time. These ultrastructural data have been confirmed in vitro by the interaction of T₃ with plasma membrane, nuclear membrane, nuclear matrix and mitochondria by real-time biospecific interaction analysis in a BIAcore^™ system. These results demonstrate that T₃ binds to hepatocytes before internalization, is transported both to mitochondria and to the nuclear envelope and translocated into the nuclear matrix.     

Localization of lipoprotein in pre- and post-transition atherosclerotic lesions following short-term incubation with [125I]LDL

Summary Ultrastructural autoradiography has been used to test the hypothesis that atherosclerotic regions of vessels differ with respect to lipoprotein uptake and localization. White Carneau pigeons, in which the prevalence and localization of aortic lesions are highly predictable, were fed a 0.25% cholesterol-supplemented diet to accelerate atherosclerosis. One hour prior to necropsy the birds were given a single intravenous injection of homologous [¹²⁵I]LDL (low-density lipoprotein). Plasma die-away and tissue distribution of label were determined, and after the birds had been killed, the aortas, spleen and liver were processed for electron microscope autoradiography. Initial [¹²⁵I]LDL uptake was rapid, with 35% of the label removed within 30 min. Predominant accumulation was in the liver, followed by the lung, kidney, the spleen and the aorta, in which the [¹²⁵I]LDL level was approximately 4% that of the liver. Autoradiographic analysis documented hepatocyte (33%) and Kupffer cell (19.9%) localization in the liver and reticuloendothelial cell (57.4%) localization in the spleen. The aortic analysis involved serially sectioned lesions for direct comparison of non-lesion, lesion/non-lesion interface (edge) and deep lesion regions. Analysis of 2275 silver grains documented a ten-fold increase in LDL accumulation at the lesion edge (as compared to adjacent non-lesion) where macrophage foam cells contained more than 70% of the label. The other 30% was distributed equally among endothelium, the intimai matrix and smooth muscle cells. This distribution changed with more complex (deeper) lesions, although grain density in the complex lesions was comparable to the edge. In the complex regions, macrophage foam cell grains were reduced to 37%, whereas smooth muscle cell (22%) and the extracellular matrix (24%) label were both increased. These studies substantiate enhanced accumulation of lipoprotein specifically at lesion sites in the aorta and demonstrate a shift from macrophage localization at the developing edge to smooth muscle cell and the extracellular matrix in more complex deeper lesions.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. I. Observations in hepatocytes

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of [3H]N-acetylmannosamine (8 mCi) and then sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the silver grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. Meanwhile, the proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h and over 55% at 4 and 24 h; two-thirds of these grains lay over the sinusoidal membrane, and the remainder were equally divided over the lateral and bile canalicular membranes. Many silver grains also appeared over lysosomes at the 4- and 24-h time intervals, accounting for 15-17% of the total. At 3 and 9 d after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling such that by 9 d only a very light reaction remained. Because our biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), the interpretation of these results is that sialic acid is incorporated into these molecules in the Golgi apparatus and that the latter then migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. It is possible that some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

Uptake of lipoproteins by in situ perfused rat ovaries: identification of binding sites for high density lipoproteins

We have examined the uptake and distribution of 125I-labeled human high density lipoprotein, apolipoprotein E-free (hHDL3), 125I-rat high density lipoprotein (HDL), and human HDL (hHDL) reconstituted with [3H]cholesteryl linoleate after their in situ vascular perfusion to ovaries of gonadotropin-primed immature rats on days 6-9 post human chorionic gonadotropin (hCG)-injection. Some rats were treated with 4-aminopyrazolopyrimidine to reduce plasma lipoproteins and ovarian cholesteryl ester stores. Perfused ovaries were analyzed biochemically and autoradiographically, and progestin content of the ovarian effluent was quantified. Infusion of ovine luteinizing hormone and hHDL increased ovarian progestin secretion severalfold, indicating that the perfused ovary was functional. After perfusion with HDL reconstituted with [3H]cholesteryl linoleate, radioactive progestin appeared in the effluent; thus, sterol carried by exogenous HDL was converted to steroid. At 37 degrees C, uptake of 125I-hHDL3 was greatest after 15 min of perfusion with label. This was decreased by 80% when the perfusion was carried out at 4 degrees C and by 70-95% when excess unlabeled hHDL, but not human low density lipoprotein (hLDL), was included in the perfusate with 125I-hHDL. Aminopyrazolopyrimidine treatment enhanced 125I-hHDL uptake twofold. After perfusion for 15 min with 125I-hHDL3, radioactivity in the ovary was high for 3-30 min of HDL-free wash, then declined 75% by 30-60 min. With light and electron microscope autoradiography, 125I-hHDL3 was localized to corpora lutea, both along luteal cell surfaces and over their cytoplasm. The plasma membrane grains appeared to be associated with segments that lacked bristle coats. Perfusion with 125I-rat HDL produced a similar pattern of labeling. In ovaries perfused with 125I-BSA, silver grains were concentrated over macrophage-like cells but were sparse over luteal cells. We conclude that the in situ perfused rat ovary takes up 125I-hHDL3 by a temperature-dependent, lipoprotein-specific process, and that this lipoprotein is accumulated by luteal cells.     

Localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus: in vitro electron microscopic autoradiographical investigation using 125I-labeled ANP

The localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus of the rat was studied by electron microscopic autoradiography using 125I-labeled ANP. A total of 1,134 silver grains counted in 30 glomeruli were distributed as follows: processes of the podocytes (36.4%), cell bodies of the podocytes (14.8%), basement membrane (12.3%), endothelial cells (6.3%), mesangial cells (7.7%) and others, for example the vascular spaces (22.5%). This finding indicates that ANP binding sites are mainly localized on the foot processes of the podocytes in the glomerulus.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

Tissue localization of [125I]triiodothyronine in the periorbital area of mice: a microautoradiographic study

A significant retention of [¹²⁵I]triiodothyronine ([¹²⁵I]T₃) in the retrobulbar orbital area of mice has been previously shown. The present study was initiated to determine tissue and intracellular localization of the thyroid hormone in the above area which is concerned in human Graves' disease of the thyroid.Male and female Balb C mice were intravenously injected with 0.1 mL of [¹²⁵I]T₃ (0.2 mCi/gmg). At various time intervals (30 s-10 min) the animals were sacrificed, bled and periorbital tissues were isolated under a dissecting microscope. Three series of samples were prepared: (a) frozen samples for cryomicrotome sections, (b) samples fixed in 10% formaldehyde for paraffin embedded tissues and (c) samples fixed in paraformaldehyde (2%), glutaldehyde (2%) and 0.1 M sodium cacodylate for embedding in Epon-Araldite-DDSA. Sections 5 μ m and 400–600 Å thick for light and electron microscopy, respectively, were coated with Ilford L4 emulsion and exposed for 9–21 days. Light microscope autoradiography demonstrated that [¹²⁵I]T₃ injected intravenously is rapidly transported in the cells of fat tissue of the peribulbar orbital area and tissues with glandular or muscular function: the hormone showed a high affinity for the intra- and extraorbital lacrymal gland cells, the cells of the Harder's gland, those of the sebaceous and meibomian glands of the eye-lids, as well as for local muscular structures. Electron microscope autoradiography showed that radioactivity is already localized inside the cells 30 s after the i.v. injection of [¹²⁵I]T₃ and it is distributed throughout the cytoplasm, with a higher concentration in the vesicles of the Harder's gland cells (rich in lipids and porphyrin), in the endoplasmic reticulum and the mitochondria of the lacrymal glands. 10 min after injection, a shifting of the radioactivity towards the nucleus area was observed. In conclusion, after _vivo injection, the thyroid hormone rapidly penetrates the cells of fat glandular and muscular tissues in the orbital area. Intracellularly, the affinity of the hormone for the secretory vesicles, rough endoplasmic reticulum, mitochondria and nucleus suggest that T₃ could play a role in secretory and metabolic functions of the tissues in the retrobulbar orbital area.     

Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex

Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.     

Synthesis and activity profiles of atrial natriuretic peptide (ANP) analogs with reduced ring size

Three deletion analogs of the atrial natriuretic peptide, [desGly-120]-ANP(103-126), [desLeu119, desGly120]ANP(103-126) and [desGly118,120,-desLeu119]-ANP(103-126), were synthesized by the solid-phase method. Successive elimination of the non-functional residues in positions 120-118 of the peptide sequence resulted in a progressive potency reduction in the rabbit aorta assay, in the bioassay monitoring suppression of aldosterone secretion from bovine zona glomerulosa cells and in a binding assay based on displacement of radioiodinated ANP(101-126) from bovine zona glomerulosa cell membranes. The fact that the deletion analogs showed quantitatively different relative potencies in each of the three in vitro assay systems was interpreted as further evidence in support of the existence of two or more classes of ANP receptors or binding sites.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.