Concentration Of Silver Grains Research Articles

Localization and Diurnal Expression of Melanopsin, Vertebrate Ancient Opsin, and Pituitary Adenylate Cyclase-Activating Peptide mRNA in a Teleost Retina

Localization and Diurnal Expression of Melanopsin, Vertebrate Ancient Opsin, and Pituitary Adenylate Cyclase-Activating Peptide mRNA in a Teleost Retina

Binding of highly concentrated maxacalcitol to the nuclear vitamin D receptors of parathyroid cells

Injection of maxacalcitol (OCT) directly into the parathyroid gland (PTG) is a clinically safe and effective treatment for advanced secondary hyperparathyroidism (A-SHPT) resistant to conventional medical treatment. In the present study, the degree of nuclear localization of directly injected OCT in parathyroid cells (PTC) was investigated by microautoradiography (mARG) in a model of A-SHPT. The 5/6 nephrectomized Sprague-Dawley rats were fed a high-phosphate and low-calcium diet for 8 weeks and consequently the level of vitamin D receptor (VDR) in their PTC severely decreased. The bilateral PTG were surgically exposed and only the left gland were directly injected with 3H-OCT (DI-3H-OCT). The time course of the changes in both radioactivity and localization of 3H-OCT in the bilateral glands was analysed using a bioimaging analyser system and mARG, respectively. A very high dose of unlabelled calcitriol was administered intravenously (IV-1,25D3) prior to DI-3H-OCT, as a competitive study. Peak radioactivity levels in the directly injected and intact PTG occured immediately and 1 h, respectively, after DI-3H-OCT, and the difference was about 50-fold higher in the treated gland. The of mARG showed a marked concentration of silver grains in the nuclei of PTC in the gland treated with DI-3H-OCT and that concentration was significantly suppressed by IV-1,25D3. Direct injection of OCT into the PTG enables the administration of the highly concentrated drug for specific binding to nuclear vitamin D binding sites, including VDR of PTC, which markedly suppresses the parathyroid hormone, improves the response to calcium and vitamin D and induces apoptosis in PTC.

Ascorbic Acid: A Precursor of Oxalate in Crystal Idioblasts ofYucca torreyiin Liquid Root Culture

Liquid‐cultured primary roots of Yucca torreyi L. (Agavaceae), which are similar to its intact roots, develop uninterrupted files of calcium oxalate crystal idioblasts with raphide bundles in their cortex, beginning just proximal to the terminal meristem. Each single file of idioblasts displays a basipetal ontogenetic sequence. [1‐14C]glycolic acid, [1‐14C]glyoxylic acid, and l‐[1‐14C]ascorbic acid, all of which are potential precursors of oxalate, were each added to different flasks that contained a sterile liquid medium and isolated roots and were allowed to interact with the roots for 45 min. After thorough washing, the roots grew for periods that extended from 1.6 h to 24 h postincorporation before being fixed for light microscope autoradiography. Autoradiography of root sections with the l‐[1‐14C]ascorbic acid at the 1.6–6.0‐h incorporation times showed concentrations of silver grains over the idioblasts, primarily over the vacuole crystal bundles and cytoplasmic plastids. The [1‐14C]glyoxalic acid– and [1‐14C]glycolic acid–labeled root sections showed a smaller amount of silver grains distributed over the entire sections, but these grains were not concentrated over the crystal idioblasts. These results strongly indicate that the l‐[1‐14C]ascorbic acid is the immediate precursor of oxalate in the crystal idioblasts of Y. torreyi primary roots and support more recent biochemical data regarding oxalate synthesis in higher plants. The use of roots in liquid culture containing uninterrupted files of developing crystal idioblasts could serve as a model system for additional biochemical, physiological, and molecular studies that seek to understand the formation and functional significance of crystal idioblasts in higher plant organs.

Relative contributions of a CVO and the microvascular bed to delivery of blood-borne IL-1alpha to the brain.

Diffusion from brain regions lacking a blood-brain barrier (BBB) and saturable transport across capillaries are possible pathways for the entry of blood-borne interleukin-1alpha into the central nervous system (CNS). To assess the involvement of these putative routes, mice received intravenous injections of radioiodinated interleukin-1alpha, and their brains were subjected to emulsion autoradiography. The resulting patterns of silver grain distribution showed that diffusion of interleukin-1alpha from the choroid plexus and the subfornical organ was greatly restricted. These restrictive properties were quantified by the determination of D1/2 values, the distances needed for the concentration of silver grains to decrease by one-half. Within several brain regions, a subset of the microvasculature indicated transport of interleukin-1alpha across the BBB. Individual microvessels showed different patterns of transport ranging from robust to absent. The high degree of containment of blood-borne interleukin-1alpha within the regions lacking a BBB indicates that these sites cannot account for total delivery of the cytokine into the brain and suggests instead that the microvascular network may serve as the major route of entry into the CNS.

DNA synthesis and mRNA accumulation during germination of embryos of the orchidSpathoglottis plicata

Germination of embryos of the orchid,Spathoglottis plicata Blume involves the formation of a protocorm in which DNA synthesis and cell divisions are confined to the proximal end whereas cells at the distal end undergo enlargement. Although3H-thymidine was incorporated into the distal cells of the embryo during the early period of germination, DNA synthesis was not followed by mitosis and cytokinesis. Poly(A)-RNA detected by in situ hybridization of sections to3H-poly-(U) was present uniformly in all cells of the embryo of the dry seed. However, coincident with the formation of the apical meristem, there was an increase in the density of auto-radiographic silver grains in the cells of the embryo, with a concentration of silver grains in the proximal part. The results indicate that regulatory events in the embryo prior to seed maturity determine the fate of its proximal and distal parts during germination.

Stability of elastin in the developing mouse aorta: a quantitative radioautographic study.

Elastic lamina growth during development and the ultimate stability of elastin in the mouse aortic media was investigated by light and electron microscopic radioautography. Following a single subcutaneous injection of L-[3,4-3H]valine at 3 days of age, animals were killed at 9 subsequent time intervals up to 4 months of age. One day after injection, radioautographic silver grains were primarily observed over the elastic laminae; however, silver grains were also seen over the smooth muscle cells and extracellular matrix. By 21 to 28 days of age, the silver grains were almost exclusively located over the elastic laminae. From 28 days to 4 months of age, the distribution of silver grains appeared relatively unchanged. Quantitation of silver grain number/micron2 of elastin showed a steady decrease in the concentration of silver grains associated with the elastic laminae from 4 to 21 days of age. After this time, no significant difference in silver grain concentration was observed. Since the initial decrease in grains/micron2 of elastin corresponds to a period of rapid post-natal growth, the decrease is likely to be a result of dilution of the radiolabel due to new elastin synthesis. With the assumption that little or no significant turnover occurs during this time, a constant growth rate of 4.3% per day was predicted by linear regression analysis. Since no significant difference in the concentration of silver grains was observed from 28 to 118 days of age, no new growth or turnover of elastin can be said to occur during this time period. This is supported by the observation that animals injected with radiolabeled valine at 28 days and 8 months of age showed no significant incorporation of radiolabel into the elastic laminae. The results from this study present the first long-term radioautographic evidence of the stability of aortic elastin and emphasize that initial deposition of elastin and proper assembly of elastic laminae is a critical event in vessel development.

Ecdysteroid binding sites localized by autoradiography in the central nervous system of precommitment fifth-stadium Manduca sexta larvae

Brains and subesophageal ganglia from day 3.5 fifth stadium larvae of Manduca sexta were incubated in vitro with 4 nM tritiated ponasterone A, a 20-hydroxyecdysone analog, to determine whether uptake and specific binding of ecdysteroids occur at a cellular level. These tissues, which were taken just prior to the commitment peak in the hemolymph-ecdysteroid titer, showed saturable uptake of 3H-ponasterone A after 40-60 min of incubation. Uptake was blocked by the addition of 400 nM unlabelled ponasterone A, or of 500 nM or 1000 nM 20-hydroxyecdysone. RH 5849, a synthetic 20-hydroxyecdysone agonist with a long half-life, for which ecdysteroid receptors have low affinity, also reduced ponasterone A uptake at a concentration of 10 microM. Autoradiographs of 4 microns sections of brains revealed distinct nuclear concentrations of silver grains over cell populations in the pars intercerebralis, pars lateralis, and ventral tritocerebrum. Nuclear labelling was also found in many small cells around the mushroom bodies and the neuropil, and between the inner and outer larval optic lobes. Nuclear labelling of cells in the subesophageal ganglion was observed in the fronto-medial and lateral regions, in small cells around the neuropil, and caudally in a few large neurons. In addition to cells with nuclear labelling, both brains and ganglia at this development stage contained cells with exclusively cytoplasmic or both nuclear and cytoplasmic labelling. None of these apparent binding sites were observed in the competition experiments, suggesting that the binding is specific.

Cellular gene expression for calbidin‐D28k in mouse kidney

Gene expression for calbindin-D28k, the 28,000 relative molecular mass vitamin D-dependent calcium-binding protein, was measured in cells of the murine nephron by in situ hybridization on tissue sections (hybridization cytochemistry). Radiolabeled (35S-UTP), single-stranded RNA complementary to calbindin-D28k-mRNA (probe RNA) was prepared from linearized cDNA template and used for the hybridizations. Autoradiography was carried out and cellular levels of hybridization signal (silver grains) were quantified. After correction for background the concentration of silver grains was more than 350% greater in the distal tubule than in either the proximal tubule or the glomerulus. The relative cellular level of mRNA in the cytoplasm, as reflected in silver grains/cell, of the distal tubules with probe RNA was 3.4 times greater than that with control RNA. Cells of the distal tubule were the only apparent sites of specific hybridization with probe RNA. The presence of calbindin-D28k-mRNA in the distal tubule corresponded to the localization of calbindin-D28k by immunocytochemistry.

The histoautoradiographic localization of taurocholate in rat liver after bile duct ligation: Evidence for ongoing secretion and reabsorption processes

To determine whether in complete obstructive cholestasis taurcholate is taken up by hepatocytes and if so whether it is secreted into bile, tritium-labelled taurocholate was localized by histoautoradiography on cryoslices from normal rat livers and from those after bile duct ligation. In non-cholestatic livers the hepatocytes of acinar zones 1 as well as the lumina and the epithelia of bile ductules and ducts became intensely labelled directly after injection of [ 3H]taurocholate into a mesenterial vein. Four hours and 4 days after bile duct ligation, hepatocytes of all three acinar zones became labelled, but in contrast to the normal state, pericanalicular concentration of silver grains was not observed, not even within 5 min. Fifteen days after bile duct obstruction, cryoslices taken 2 min after injection of [ 3H]taurocholate exhibited an intense silver grain labelling of all acinar zones, with the highest density at bile canalicular areas of the liver cell plates as well as the proliferated bile ductules and bile ducts. The biliary epithelium of small bile ductules and ducts of non-cholestatic and of bile duct-obstructed livers were also covered with silver grains; the epithelium of larger ducts exhibited significant labelling predominantly at the lateral sites of the celis. The biliary epithelium of the common bile duct was not significantly labelled. The results indicate that in complete obstructive cholestasis (a) taurocholate continues to be taken up from blood by hepatocytes and secreted into bile, but in terms of varying duranon of obstruction, (b) all acinar zones are involved in bile salt transport, (c) in the initial phase (4 h and 4 days respectively after bile duct obstruction) hepatocytes fail to concentrate taurocholate at the canalicular site, (d) in a consecutive phase, in which bile ductules and ducts proliferate (demonstrated for a 15-day cholestasis), the taurocholate concentration at the canalicular site of hepatocytes is re-established and biliary secretion seems to be enhanced, (e) the biliary epithelium of bile ductules and ducts may play a significant role in the reabsorption and/or regurgitation of bile salts from bile to blood. Reabsorption/regurgitation of biliary constituents may also be operative in the non-cholestatic state but may become significantly enhanced with bile ductular proliferation.

Studies on the influx of [ 3H]-histidine and 45Ca through a surgical opening to rat incisor ameloblasts and adjacent enamel

Considerable controversy exists about the role of ameloblasts in transport of calcium to mineralizing enamel. The rate and pattern of incorporation of calcium and an enamel precursor (histidine) were studied autoradiographically by introducing the isotopes through a surgically created defect in the lower border of the rat mandible. Influx of the isotopes to both secretory and maturation ameloblasts and adjacent enamel was examined in large survey sections of the entire incisor at intervals of 3 min to 2 h. Substantial concentrations of silver grains were observed over both secretory and maturation ameloblasts within 5 min of placement of either isotope. 45Ca was also present in secretory and maturation enamel within this short time. The overall patterns of influx and uptake of both precursors were similar to those found when such isotopes have been administered parenterally. The amount of influx of 45Ca across secretory, smooth-ended, and ruffle-ended maturation ameloblasts was compared qualitatively and found to be similar. The reproducibility of this surgical technique was demonstrated as well as its usefulness in combination with survey sections for multi-method investigations of rat incisor enamel formation and mineralization.

Localization of vasopressin-, vasoactive intestinal polypeptide-, peptide histidine isoleucine- and somatostatin-mRNA in rat suprachiasmatic nucleus.

Messenger RNAs (mRNA) coding for vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), somatostatin and vasopressin were localized in the suprachiasmatic nucleus (SCN) of the rat hypothalamus using in situ hybridization histochemistry. Specific mRNA coding for each of these peptides was distributed in areas coextensive with the immunohistochemical localization of the appropriate peptide. The autoradiographic signal produced with probes to VIP and PHI created dense concentrations of silver grains over neuronal perikarya in the ventrolateral SCN, and the coextensive distribution of both VIP- and PHI-mRNAs suggests that both peptides are synthesized within the same neurons. The distribution of somatostatin-mRNA was distinct from the of VIP and PHI. Labeled neurons are observed at the interface of the two SCN subdivisions and the distribution of these neurons is identical to those shown to contain somatostatin immunoreactivity. Vasopressin-mRNA is also differentially concentrated within neurons in the dorsomedial subdivision of the SCN in an area that is coextensive with vasopressin-immunoreactive perikarya. The discrete pattern of hybridization for each of these mRNAs indicates that each of these peptides are synthesized in SCN neurons and reaffirms the differential distribution of each of these chemically defined cell populations within cytoarchitecturally distinct subdivisions of the nucleus.

Sex steroid receptors in the stomach, liver, pancreas, and gastrointestinal tract of the baboon

Sex steroids have been shown to have a marked effect on the physiologic activities of the liver and the gastrointestinal tract. We performed autoradiographic studies using [3H]estradiol and [3H]dihydrotestosterone on male and female baboons for the purpose of identifying estrogen or androgen receptors, or both, in the liver, pancreas, stomach, and small and large intestines of baboons. Evidence for the presence of estrogen and androgen receptors was made apparent by high concentrations of silver grains over the nuclei of the cells of these tissues. Androgen receptors were largely confined to the nuclei of the smooth muscle cells of the tunica muscularis of the gut wall and the connective tissue interstitial cells of the liver, pancreas, stomach, and intestines. Estrogen receptors were prominent in the nuclei of the vascular smooth muscle cells in the liver, pancreas, gut, and the majority of the endocrine islet cells. These observations suggest that a variety of different cell types of the liver, pancreas, and gastrointestinal tract contain estrogen and androgen receptors that might modulate their cellular activities and influence several different physiologic processes.

Renal handling and effects of [3H]digoxin and interactions with quinidine in the avian kidney.

Renal handling and effects of tritium digoxin and interactions with quinidine in the avian kidney were studied using a modified Sperber technique. Results showed that tritium digoxin was extracted at the peritubular side of the nephron in a process competitively inhibited by increasing amounts of unlabelled digoxin. Light microscope autoradiography showed distinct concentrations of silver grains only over distal tubules in the injected kidney. Inhibition of the proximal tubular transport systems for organic anions and cations, respectively, did not change extraction. Addition of quinidine to the injection solution up to an estimated concentration of 1.4 X 10(-5) M in systemic blood significantly lowered 1 min peritubular extraction of tritium digoxin. However, when the amount of quinidine was further increased, extraction of tritium digoxin augmented significantly. Tritium recovery in urine after renal portal bolus injection of tritiated and unlabelled digoxin already showed a distinct ipsilateral peak 2 min after injection with an equally distinct peak of ipsilateral sodium excretion appearing 1 min later. When quinidine was added to the bolus ipsilateral tritium recovery in urine (0-7 min) was halved, with the true tubular excretion fraction (TTEF) lowered by two-thirds, but without changes in the magnitude of ipsilateral natriuresis. Contralateral natriuresis increased more than four-fold with quinidine in the bolus in spite of unchanged tritium recovery in the urine. Thus, our results show tritium digoxin to be extracted from peritubular blood through a specific process probably localized to the distal nephron of the avian kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of [3H] thymidine incorporation differ in immature rats and mature, cycling rats.

By the time follicular development has progressed to the preovulatory stage, granulosa cells abutting the basement membrane no longer incorporate [3H] thymidine (3H-TdR). The purpose of this experiment was to determine when, during the course of follicular growth, cell proliferation in these mural granulosa cells ceases. Autoradiographs were prepared following continuous 3H-TdR infusion in vivo, or incubation with 3H-TdR in vitro. In cycling rats, the concentration of silver grains over mural regions of the granulosa layer was lower than over antral regions of most follicles with greater than 1000 cells in the largest cross section (LCS). This centripetal labeling pattern became more striking as follicular size increased. By proestrus, only the cells of the discus proligerus (cumulus and the portion of the follicular wall supporting the cumulus oocyte complex) continued to incorporate 3H-TdR. In contrast to cycling rats, centripetal labeling patterns were not seen in ovaries of prepubertal rats, even in follicles of the same size. The difference in follicular growth patterns between these two types of animals suggests an influence of cyclic gonadotropin surges on the control of granulosa cell proliferation.

Columnar organization and reciprocity of commissural connections in cat primary auditory cortex (AI).

The laminar distribution and reciprocity of commissural axon terminals and cells of origin in cat primary auditory cortex (AI) were studied after injections of tritiated proline combined with horseradish peroxidase in the middle ectosylvian gyrus. Terminal fields were found in every cortical layer in the contralateral AI, and they were characterized quantitatively. The largest concentration of silver grains was in layer III (about 25% of the total number of silver grains) and, to a lesser extent, in layers V, VI, and I (some 18% of the total in each layer). The labeling in layer I was concentrated in its deeper half, while the labeling in the other layers was more homogeneous. Layer IV had the least labeling, followed by layer II, each receiving about 10% of the total. The labeling was always heaviest over the neuropil and lightest over neuronal perikarya. Commissural terminal fields formed radial patches oriented perpendicularly to the pia, and averaging 543 micron in width. There was consistently three times more silver grains in a patch than in an inter-patch area. However, the number of silver grains in an inter-patch area was always significantly above background, indicating a possible commissural projection to these zones as well. The patches of commissural terminal fields formed bands oriented across AI and running in a caudoventral to rostrodorsal direction. Strict reciprocity between the commissural cells of origin and terminal fields was not found at the light microscopic level when adjacent sections, corrected for differential shrinkage, were compared. Often, patches of terminal fields were free of retrogradely labeled cells and, conversely, there were patches of labeled cells without an overlying commissural terminal field. The terminal fields connected homotopic regions of the contralateral AI, and every region of AI received commissural innervation, unlike the primary somatic sensory and visual cortex, where large zones receive only a few commissural afferents. The more complete pattern of interhemispheric connectivity in auditory cortex is in contrast to the less continuous commissural representation in other sensory neocortical fields. Perhaps this pattern contributes to the anatomical representation of binaurality in auditory cortex.

Localization of the lipid intermediate pathway of protein glycosylation in oviduct cell types

Oviduct tissue slices were incubated with [ 3H]-leucine or [ 3H]-mannose in the presence and absence of tunicamycin, a specific inhibitor of lipid-mediated protein glycosylation. Conditions were established where tunicamycin had maximal effect on [ 3H]-mannose incorporation (>90% inhibition) but a minimal effect on [ 3H]-leucine incorporation (<10% inhibition) into total TCA-insoluble products. Analysis of incubated tissues by SDS-polyacrylamide gel electrophoresis revealed that in the absence of tunicamycin, [ 3H]-mannose was incorporated into only a few proteins, of which ovalbumin represented the major radiolabeled component. Tunicamycin markedly reduced the incorporation of [ 3H]-mannose into ovalbumin and other oviduct glycoproteins. In contrast, analysis by SDS-polyacrylamide gel electrophoresis showed that [ 3H]-leucine was incorporated into a variety of proteins in the absence of tunicamycin. The radioactivity profile of some of these proteins was shifted toward lower M r when oviduct slices were incubated in the presence of tunicamycin, with only a minimal decrease in protein labeling. Light microscopic autoradiograms of tissue incubated with [ 3H]-leucine in either the presence or absence of tunicamycin exhibited extensive labeling of tubular gland and epithelial cells. In the absence of tunicamycin, these cell types also become markedly labeled with [ 3H]-mannose; however, incorporation of label in both cell types was substantially reduced in the presence of tunicamycin. Qualitatively, labeling of tubular gland cells appeared greater than that of epithelial cells, largely due to the concentration of silver grains over the dense population of secretory vesicles in the tubular gland cells. Connective tissue (stromal) cells were not labeled above background levels with either radiolabeled substrate, whereas the endothelial cells of the vascular elements which intermittently permeate the oviduct were moderately labeled with both [ 3H]-leucine and [ 3H]-mannose. We conclude that the tubular gland cells represent the primary contributors to the overall activity of the lipid intermediate pathway in the oviduct.

Peripheral-type benzodiazepine receptors in endocrine organs: autoradiographic localization in rat pituitary, adrenal, and testis.

We have used [3H]Ro5-4864, a ligand selective for peripheral-type benzodiazepine receptors, to identify and localize peripheral-type benzodiazepine receptors in endocrine organs. Autoradiographic studies reveal an uniform distribution of [3H]Ro5-4864 binding sites within the anterior, intermediate, and posterior lobes of the pituitary gland, with highest concentrations present in the posterior pituitary. In rat adrenal gland, specific binding sites for [3H]Ro5-4864 are found only in the adrenal cortex, with highest density in the zona glomerulosa and significantly lower concentrations in the zona fasciculata and zona reticularis. [3H]Ro5-4864-associated silver grains in the testis are intensely localized over the interstitial tissue; low concentrations of silver grains are present over the epithelium of the seminiferous tubules but are absent from the tubular lumen. These studies demonstrate a differential and discrete localization of peripheral-type benzodiazepine receptors in rat pituitary, adrenal, and testis.

Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.

Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [3H]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.

Distribution of newly formed hepatic glycogen in adrenalectomized rats as observed by radioautography after injection of 3H-galactose

In order to study early events of glycogen synthesis, we have used adrenalectomized (ADX) rats fasted overnight and injected with the synthetic glucocorticoid dexamethasone (DEX) to stimulate glycogen synthesis. Rats were given DEX 0-5 hr prior to sacrifice and injected with 2 mCi 3H-galactose 1 hr prior to sacrifice. Liver was prepared for light (LM) and electron microscopic (EM) radioautography by routine procedures.The concentration of silver grains over hepatic cytoplasm was measured in LM radioautographs using a Zeiss Videoplan. The hepatocytes were categorized as unlabeled if no silver grains (gr) were present, lightly labeled (&lt;10gr/100 μm2 cytoplasm) or intensely labeled (&gt;10 gr/1002 μm cytoplasm). Although very few hepatocytes showed heavy labeling after 1 hr treatment with DEX, by 2 hr after DEX treatment 8% of the cells distributed throughout the lobule were intensely labeled.

Acquisition of multiple nuclei and the activity of DNA polymerase alpha and reinitiation of DNA replication in terminally differentiated adult cardiac muscle cells in culture.

Terminally differentiated ventricular cardiac muscle cells isolated from the adult rat and maintained in cell culture have been observed to acquire multiple nuclei. In one cultured myocyte as many as 10 nuclei have been counted. Apparently, these multiple nuclei are formed by DNA replication followed by karyokinesis; the cells must then fail to complete mitosis and divide. To investigate whether DNA synthesis was occurring, the cells were cultured in the presence of [3H]thymidine and then processed for autoradiography. Mononucleated, binucleated, and multinucleated cells incorporate [3H]thymidine into DNA as evidenced by the high concentration of silver grains over their nuclei. Peak periods of incorporation were observed to occur at 10- to 12-day intervals; at 11, 23, and 33 days after initially placing the cells in culture. When the cells were maintained in the presence of [3H]thymidine continuously from Day 7 to Day 17 of culture, 23% of the cells became labeled. If the cells were cultured continuously for 30 days in the presence of [3H]thymidine, from Day 10 to Day 40, 56% of the cells were labeled. Isopycnic gradient analysis indicates that this thymidine incorporation was into DNA that was being replicated semiconservatively; these experiments did not eliminate the possibility, however, that this incorporation was due to amplification of specific genes, such as those coding for the contractile proteins. The activity of DNA polymerase alpha also returns to these cells. These studies demonstrate that the terminally differentiated mammalian ventricular cardiac muscle cell, previously thought to have permanently lost the capacity to replicate DNA during early development, is able to reinitiate semiconservative DNA replication when grown in culture.