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Biomedical subjects

S Malamed

Publications and source records attributed to S Malamed.

At least 37 records · Page 2Linked to original sources
PubMed1987

A morphometric analysis of adrenocortical actin localized by immunoelectron microscopy: the effect of adrenocorticotropin.

The localization of actin and the effect of ACTH on its concentration was examined in freshly isolated rat adrenocortical cells. Lowicryl K4M-embedded cells were used for the immunoelectron localization of actin; gold was used as a label for immunoreactive sites. Actin was at least 4 times as concentrated at the cortical cytoplasm as in the lipid droplets and at least 5 times as concentrated in the microvilli as in the lipid droplets. ACTH stimulation approximately doubled the concentration of actin in the cortical cytoplasm and increased by 50% the concentration of actin in the microvilli. The microvillar contribution to the cell surface area was 40% higher in ACTH-stimulated cells than it was in unstimulated cells. These results provide quantitative evidence suggesting that actin and the microvilli participate in steroid secretion by the adrenocortical cell.

Actins↗
PubMed1986

Progesterone and testosterone production by dispersed rat placental cells.

Isopycnic separation and unit gravity sedimentation were employed to identify the rat placental cell types capable of producing progesterone and testosterone. Subdivision of Day 12-dispersed placental cells in Percoll gradients revealed that fractions (less than 1.048 g/ml) containing giant cytotrophoblast cells produced greater quantities of progesterone (p less than 0.01) than did fractions (greater than 1.048 g/ml) with equal numbers of placental cells but void of giant cytotrophoblasts. Unit gravity sedimentation of Day 16-dispersed placental cells revealed that when incubated, isolated giant cytotrophoblast cells were capable of producing both progesterone and testosterone. Both of the separation studies strongly suggested that other cell types also produce steroids. However, the biosynthetic capacity of the giant cytotrophoblast cell appeared to be 1000-fold greater than that of the other cell types. Incubation of Day 12-dispersed placental cells with human chorionic gonadotropin or 3',5'-cyclic adenosine monophosphate did not further increase progesterone production as compared to untreated control incubates, suggesting rat placental steroidogenesis is not under trophic hormone control. Electron microscopic observations of giant cytotrophoblast cells revealed a complex ultrastructure suggesting a variety of physiological functions.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

Actin Cytoskeleton↗
PubMed1985

An electron microscopic study of terminals of rapidly adapting mechanoreceptive afferent fibers in the cat spinal cord.

The intra-axonal horseradish peroxidase technique was used to examine the central terminals of 7 A beta primary afferent fibers from rapidly adapting (RA) mechanoreceptors in the glabrous skin of the cat's hindpaw. At the light microscopic level, labelled collaterals were seen to bear occasional boutonlike swellings, mostly (75-82%) of the en passant type. These swellings were distributed more or less uniformly from lamina III to a dorsal part of lamina VI in the dorsal horn, over a maximum longitudinal extent of about 4 mm. At the electron microscopic level, we observed that labelled boutons of RA afferent fibers were 1.0 to 3.3 micrometers in longest sectional dimension, and contained clear, round synaptic vesicles. They frequently formed asymmetric axospinous and axodendritic synapses and commonly appeared to receive contacts from unlabelled structures containing flattened or pleomorphic vesicles plus occasional large dense-cored vesicles. The examination of synaptic connectivity over the entire surface of individual boutons indicated that RA afferent boutons each made contacts with an average of one spine and one dendrite and, in addition, appeared to be postsynaptic to an average of two unlabelled vesicle-containing structures. This synaptic organization was, in general, more complex than that we had seen previously in Pacinian corpuscle (PC) and slowly adapting (SA) type I mechanoreceptive afferent fibers. Our findings indicate that RA, SA, and PC afferent terminals, while displaying some differential synaptic organizations, have many morphological and synaptological characteristics in common. These afferent terminals, in turn, seem to be generally distinguishable from the terminals of muscle spindle Ia afferents or unmyelinated primary afferents.

Animals↗
PubMed1985

Isolated adrenocortical cells of the domestic fowl (Gallus domesticus): steroidogenic and ultrastructural properties.

Isolated adrenocortical cells from White Leghorn chickens (Gallus domesticus) were compared to those from rats (Rattus norvegicus). Cells were prepared from collagenase-dispersed adrenal glands of sexually mature male animals. Corticosterone was measured by radioimmunoassay after incubation for 2 h with steroidogenic agents. Of the four ACTH analogues used, three were 6-17 times more potent with rat cells than with fowl cells (potencies were indicated by half-maximal steroidogenic concentrations). However, 9-tryptophan (O-nitrophenylsulfenyl) ACTH was 8 times more potent with fowl cells than with rat cells, thus suggesting that ACTH receptor differences exist between the two cell types. In addition, cAMP analogues were 10 times more potent with rat cells than with fowl cells suggesting that fowl corticosteroidogenesis is less dependent on cAMP than is rat corticosteroidogenesis. At equal cell concentrations, rat cells secreted 20-40 times more corticosterone than did chicken cells when they were maximally stimulated. Although rat cells converted 8 times more pregnenolone to corticosterone than did fowl cells, the half-maximal steroidogenic concentration for pregnenolone-supported corticosterone synthesis was the same for both cell types (about 5 microM). This suggests that fowl cells have lower steroidogenic enzyme content rather than lower steroidogenic enzyme activity. An unusual feature seen in the isolated fowl adrenocortical cells was an abundance of intracellular filaments.

Adrenal Cortex↗
PubMed1985

Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl.

Maturation of domestic fowl corticosteroidogenesis was evaluated using purified adrenocortical cells. Basal corticosterone production decreased steadily from 2 days to 26 weeks after hatching. However, maximally stimulated corticosterone production was not changed. In contrast, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH analogs increased approximately 40 times by 26 weeks, but the ED50 values of 8-bromo-cyclic AMP and pregnenolone were not changed. This suggests that adrenocortical cell sensitivity to ACTH decreases with maturation of the domestic fowl.

Adrenal Cortex↗
PubMed1984

Ultrastructure of pacinian corpuscle primary afferent terminals in the cat spinal cord.

The glabrous skin of the hindlimb of the cat contains 3 types of low-threshold mechanoreceptors: Pacinian corpuscles (PC), and slowly and rapidly adapting receptors. In the present study, 12 primary afferent fibers transmitting impulses from PC were injected intra-axonally with horseradish peroxidase (HRP) in the spinal cord to examine the morphology of their terminals in the dorsal horn. At the light microscopic level, terminal arborizations were observed in laminae II-VI of the dorsal horn, extending up to 7 mm rostrocaudally in and near the seventh lumbar segment. Bouton-like swellings, predominantly (67%) of the en passant type, were distributed in two discrete clusters, one concentrated rostrally in Rexed's laminae III-IV, and the other concentrated caudally in lamina V. At the electron microscopic level, a combination of morphometric and serial reconstructive analyses with 3 fibers revealed the following. Boutons labelled with HRP invariably contained clear round vesicles, approximately 40 nm in diameter. Labelled bouton sections had longest dimensions of 1.84 +/- 0.63 micron. Their shapes varied from rounded to elongated forms with occasional scalloped appearances. A majority (73%) of the contacts associated with HRP-filled boutons were made with dendritic spines and shafts. Thick postsynaptic densities were usually associated with these synapses, although thinner densities were also observed. 24% of the contacts made by labelled boutons were synapse-like contacts with unlabelled vesicle-containing structures. The vesicles in the unlabelled structures were usually pleomorphic, but sometimes round. These contacts were identified as 'synapse-like' because labelling obscured possible landmarks necessary for definitive identification of synapses. However, in most of these contacts, there was an accumulation of vesicles near the cleft on the unlabelled side, suggesting that the labelled boutons were postsynaptic. Only 3% of the contacts made by labelled boutons were axosomatic. The lengths of contacts with dendritic spines (0.49 +/- 0.23 micron) or with dendrites proper (0.45 +/- 0.20 micron) were significantly longer than those with vesicle-containing unlabelled structures (0.31 +/- 0.18 micron). The portions of cross-sectional bouton contours devoted to synaptic or synapse-like contacts accounted for 9-13% of the perimeters. The larger the bouton, the longer the summed lengths of contacts. Serial reconstruction of selected labelled boutons revealed both simple and quite complex synaptic organizations, including glomeruli with labelled boutons as the central component.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1984

Self-suppression of corticosteroidogenesis: evidence for a role of adrenal 5 alpha-reductase.

Exogenous corticosterone (B), the natural glucocorticoid product of rats, suppressed endogenous B production of isolated rat adrenocortical cells induced by alpha ACTH-(1-24), [9-tryptophan (O-nitrophenylsulfenyl)]ACTH-(1-24) [( Trp (Nps)9]ACTH-(1-24], and cAMP as well as pregnenolone supported-steroidogenesis. This self-suppression occurred within 2 h. It was dependent on the concentration of exogenous B. However, self-suppression did not alter the half-maximal steroidogenic concentration (ED50) of each steroidogenic agent. In addition, exogenous B did not suppress ACTH-induced cAMP production or gross protein synthesis, as measured by leucine incorporation into bulk cellular proteins. These results with isolated cells suggested at least two mechanisms for self-suppression: 1) exogenous B inhibited steroidogenic steps in a noncompetitive manner, and/or 2) exogenous B induced B degradation. In this study we examined the effect of exogenous B on the degradation of B. Accordingly, we measured the adrenal 5 alpha-reductase activity (5 alpha RA) of cell homogenates prepared from treated cells. Isolated adrenocortical cells were incubated for 2 h with alpha ACTH-(1-24), ovine PRL (oPRL), and B. They were then homogenized and assayed for 5 alpha RA, as indicated by the disappearance of exogenous B, as shown by RIA. In addition, the percentage of exogenous tritium-labeled B [( 3H]B) converted to 5 alpha-dihydrocorticosterone (DHB), the principal reduced metabolite of B, was determined by TLC. Isolated adrenocortical cells from intact rats showed insignificant 5 alpha RA and DHB formation when incubated with or without alpha ACTH-(1-24) and with or without oPRL. However, with exogenous B, there was significant 5 alpha RA and DHB formation. oPRL plus B decreased DHB formation. The effects of B and oPRL were more demonstrable with cells from hypophysectomized rats. These cells exhibited high 5 alpha RA and DHB formation; exogenous B increased these values, whereas oPRL acutely reversed the effects of hypophysectomy and exogenous B. In other work avoiding cell homogenization, exogenous B suppressed ACTH-induced B accumulation and increased DHB formation in intact cell suspensions from intact rats and intact male domestic fowl. Furthermore, exogenous B increased the conversion of [3H] pregnenolone to DHB in intact cell suspensions from intact rats, showing that B synthesized de novo as well as exogenous B can be degraded during self-suppression. These data indicate that acute self-suppression of corticosteroidogenesis is at least partly mediated by an increase in 5 alpha RA.

Adrenal Cortex↗
PubMed1983

An electron microscopic study of primary afferent terminals from slowly adapting type I receptors in the cat.

Primary afferent fibers transmitting impulses from slowly adapting (SA) Type I receptors in the glabrous skin of the hind paw of the cat were injected intraaxonally in the spinal cord with horseradish peroxidase (HRP). At the light microscopic level, terminal arborizations were observed in the medial dorsal horn extending up to 6 mm rostrocaudally in and near the seventh lumbar segment. Boutonlike swellings labelled with HRP were distributed in clusters in Rexed's laminae III-VI. There was a tendency for the most dorsal clusters from an individual fiber to be located rostrally and for the most ventral clusters to be located caudally. At the electron microscopic level, a combination of morphometric analysis and serial reconstruction revealed the following: (1) All the boutons labelled with HRP contained predominantly clear, round synaptic vesicles, 40-50 nm in diameter. (2) Labelled boutons (n = 75) had cross-sectional longest dimensions of 1.72 +/- 0.53 micron (Mean +/- S.D.), perimeters of 4.95 +/- 1.52 micron, and areas of 1.18 +/- 0.59 micron 2. Their shapes in section varied from rounded to elongated forms. (3) The sizes of labelled boutons decreased significantly and linearly with depth from lamina IV to VI. The shapes of the bouton cross sections also became rounder with depth in the dorsal horn. (4) About 72% of synaptic contacts associated with HRP-filled boutons were with dendritic spines and shafts; most of these synapses were of the asymmetric type. (5) The remainder (28%) of the appositions were synapselike contacts between labelled boutons and unlabelled structures containing flattened or pleomorphic vesicles, and occasional dense-cored vesicles. (6) We observed no unequivocal axosomatic contacts made by labelled boutons. (7) The lengths of synaptic appositions with dendritic spines (0.46 +/- 0.20 micron) or with dendritic shafts (0.51 +/- 0.18 micron) were significantly greater than the synapselike contacts with vesicle-containing unlabelled structures (0.29 +/- 0.09 micron). (8) Complex neuropilar organization was occasionally seen with labelled boutons as central elements, although simpler organizations were much more common. In summary, HRP-labelled fibers ended predominantly in boutons containing clear, round vesicles forming axospinous and axodendritic synapses.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Glucocorticoid control of steroidogenesis in isolated rat adrenocortical cells.

The role of end-product glucocorticoids in the regulation of corticosteroidogenesis in isolated adrenocortical cells was investigated. Trypsin-isolated cells from male rat adrenal glands were incubated with or without corticotropin (ACTH) and with or without corticosterone. Endogenous corticosterone production was determined by radioimmunoassay at the end of incubation. Cessation of ACTH-induced corticosterone production was apparent after 2-4 h of incubation. The suppression occurred later with lower cell concentrations. Corticosterone production was partially restored after washing the suppressed cells. Supernatant fluid from suppressed cell suspensions also suppressed steroidogenesis of a fresh population of cells. However, the suppressing property of the supernatant fluid was abolished after the removal of corticosterone by charcoal-dextran treatment, suggesting that corticosterone or other steroids caused the suppression. Exogenous corticosterone induced suppression over a wide range of ACTH concentrations, but did not change the half-maximal steroidogenic concentration of ACTH, indicating that the suppression does not change the sensitivity of the cells to ACTH. Suppression occurred within 30-60 min after corticosterone had been added to the incubation medium either at the start of incubation or while steroidogenesis was in progress. Suppression varied directly with the concentration of exogenous corticosterone. These data indicate that glucocorticoids can directly and acutely suppress corticosteroidogenesis and thus control adrenocortical function in concert with other regulators such as ACTH and Ca2+.

Adrenal Cortex↗
PubMed1983

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity.

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.

Adrenal Cortex↗
PubMed1983

Aging of the rat adrenocortical cell: response to ACTH and cyclic AMP in vitro.

To study intrinsic age-related changes in adrenocortical steroid production, cells isolated from rats of different ages (3 to 24 months) were used. Acute (2 hour) corticosterone production in response to stimulation by adrenocorticotrophic hormone (ACTH) and adenosine 3':5'-cyclic monophosphate (cAMP) was measured by radioimmunoassay. With age, adrenocortical cells lose much of their ability to produce corticosterone in the absence or presence of ACTH or cAMP. The loss is progressive from 6 to 24 months of age. Analysis of the data suggests that from 6 to 12 months, an intracellular steroidogenic lesion develops; in addition there may be a loss in ACTH receptors on the plasma membrane. After 12 months these defects increase and are accompanied by a decrease in receptor sensitivity to ACTH.

Adrenal Cortex↗
PubMed1982

Acute inhibition of corticosteroidogenesis by inhibitors of calmodulin action.

To identify the possible role of calmodulin in ACTH function, we tested the ability of chlorpromazine (CP) and other calmodulin antagonists to inhibit steroidogenesis of isolated adrenocortical cells of the rat. CP reversibly inhibited maximal ACTH-induced corticosterone (B) production. The presence of the drug did not alter the ED50 of ACTH stimulation (3.2 X 10(3) pg/ml), suggesting that it inhibited ACTH-induced steroidogenesis in a noncompetitive manner. The CP concentration required for half-maximal inhibition was 8.2 microM, a value close to the dissociation constant of the CP-calmodulin complex (5.3 microM). Concentrations greater than 40 microM resulted in complete inhibition. Similar concentrations of CP inhibited ACTH-induced cAMP accumulation in a dose-dependent manner, indicating an effect of the drug on early events in ACTH action. In addition, CP also apparently acted at a site distal to the point of cAMP formation, as shown by the finding that it inhibited cAMP-induced B production. CP inhibition of ACTH-induced B production was independent of the Ca2+ concentration, suggesting that the drug did not compete with Ca2+ directly. Concentrations of CP greater than 20 microM inhibited protein synthesis as measured by leucine incorporation into cellular proteins. Thus, although the inhibitory effect of high concentrations of CP on steroidogenesis might be explained by an effect on protein synthesis, the inhibition seen at 10 microM appeared to be independent of protein synthesis. Other antagonists of calmodulin action inhibited maximal ACTH-induced B production with the following relative potencies: trifluoperazine greater than CP greater than haloperidol greater than chlordiazepoxide. This order is similar to that reported for inhibition of calmodulin-activated phosphodiesterase and for binding to calmodulin. These findings suggest that calmodulin may modulate the effect of ACTH on steroidogenesis at multiple sites.

Adrenal Cortex↗
PubMed1981

Electron microscopic observations of terminals of functionally identified afferent fibers in cat spinal cord.

Using the method of intra-axonal injection of horseradish peroxidase, functionally identified afferent fibers from three slowly adapting (Type I) receptors and one Pacinian corpuscle in the glabrous skin of the hind paw of the cat were stained. Electron microscopic observation of the terminals of these fibers revealed predominantly axodendritic asymmetric synapses containing round, clear vesicles. Multiple synapses on a single dendrite were observed, separated by as little as 900 mm from one another.

Afferent Pathways↗