A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.     

Relationship between chromaffin cells and blood vessels in the rat adrenal medulla: a transmission electron microscopic study combined with blood vessel reconstructions

The rat adrenal medulla architecture was examined using a combination of medullary blood vessel reconstructions and transmission electron microscopy. The peripheral radicles of the central vein and the medullary capillaries of the medullary arteries were thus precisely identified in the electron microscopic observations. The observations confirmed that the peripheral segments of the central vein were sinusoidal vessels with an attenuated and fenestrated endothelial wall. No ultrastructural differences were observed between segments lined by epinephrine-storing cells and those lined by norepinephrine-storing cells. The findings suggest that these peripheral segments of the adrenal central vein were sites of cortical hormonal effects on the adrenal medulla. The vessel structure does not support the hypothesis that medullary chromaffin-cell development is controlled by selective distribution of adrenal blood vessels.     

Partial characterization of neuropathy target esterase and related phenyl valerate esterases from bovine adrenal medulla

The mechanism by which organo-phosphorus-induced delayed polyneuropathy is induced relates to the specific inhibition and subsequent modification (“aging”) of a protein known as neuropathy target esterase (NTE), operatively defined as paraoxon-resistant and mipafox-sensi-tive phenyl valerate (PV) esterase activity. This protein has fundamentally been investigated in hen brain, the latter being the habitually employed OPIDP study model. In the present article, a partial characterization is made of the NTE and other related PV esterases in the bovine adrenal medulla and brain; NTE sensitivity to the neurotoxic or-ganophosphorus compound mipafox is investigated, and its subcellular distribution is studied. The NTE activity of the adrenal medulla was found to be the highest of those among the tissues studied to date (5000 ± 1400 mU/g tissue; ± SD, n = 12). This activity represented 93% of the PV esterase activity resistant to 40 μm paraoxon in the par-ticulate fraction of the adrenal medulla and approximately 50% of total PV esterase activity. In the bovine brain, these proportions were 72 and 26%, respectively, i.e., similar to those described in hen brain. The mipafox inhibition curve of PV esterase activity resistant to 40μM paraoxon in the particulate fraction of the adrenal medulla suggests that NTE activity fundamentally comprises a mipafox-sensitive component with an I ₅₀ of 6.39 μM at 30 minutes, which is similar to the value reported in hen brain. NTE activity in the bovine adrenal medulla is almost exclusively limited to the particulate fraction, the microsomal fraction, plasma membrane, and chromaffin granule-enriched fractions being the highest in terms of specific activity. On the contrary, the mitochondria-enriched fraction was very poor in such activity. In bovine brain, most NTE activity was likewise limited to the particulate fraction.     

Identification and subcellular localization of catalase activity in bovine adrenal medulla and cortex

Catalase activity was detected in homogenates of bovine adrenal cortex and medulla. Analysis by equilibrium density centrifugation in isoosmotic metrizamide-sucrose gradients revealed that 70% of the medullary catalase activity was soluble while most of the remainder was found in a particulate form with a density of 1.175 g/ml. This was distinct from the densities of lysosomes, mitochondria, and chromaffin granules. Catalase activity in adrenal cortex was primarily (90%) soluble with only 6% being particulate, with a density of 1.185 g/ml. d-Amino acid, uric acid, and α-hydroxyacid oxidase activities, often associated with peroxisomes in other tissues, were absent from homogenates and catalase-containing gradient fractions from either cortex or medulla. There was an indication that some catalase activity was associated with chromaffin granules on the basis of density gradient analysis of both medullary homogenates and crude granule preparations. When granule fractions were subjected to osmotic shock, catalase activity distributed between soluble and sedimentable fractions differently from epinephrine and dopamine β-hydroxylase activity. The sedimentable catalase activity remained associated with chromaffin granule membranes upon isopycnic centrifugation. We concluded that catalase activity in both adrenal cortex and medulla was largely cytoplasmic, but that both tissues contained at least some catalase in dense organelles. Catalase activity which may be associated with chromaffin granules represents a small fraction of the total activity in the medulla.     

Effect of Oxotremorine on Ornithine Decarboxylase Activity of the Adrenal Gland in Rat

Abstract: In this work we have studied the mechanism for the increase of adrenal ODC (ornithine decarboxylase, EC 4.1.1.17) activity provoked by oxotremorine, a muscarinic agonist. 1. Oxotremorine increased medullary ODC activity maximally at 2 h. Cortical enzyme responded much more slowly. 2. Blockade of peripheral muscarinic receptors with methylatropine partially reduced the response to oxotremorine in the medulla, but not cortex. 3. Hy-pophysectomy abolished the cortical, but not the medullary, responses to oxotremorine. Methylatropine reduced the effect of oxotremorine on medullary ODC in hypophysectomized rats. 4. In unilaterally splanchnicotomized rats oxotremorine caused an increase of ODC activity of the denervated adrenal gland relative to control value; activities in both medulla and cortex were significantly lower than those observed in the innervated gland. Evidence was obtained for a compensatory increase of ODC activity of the adrenal cortex (but not medulla) on the intact side of unilaterally operated rats. 5. Surgical intervention, in the form of a sham operation for transection of the spinal cord, leads to an increase of ODC activity in both parts of the adrenal gland. Transection of the cord attenuates these increases. 6. The additional increase of medullary ODC activity owing to the administration of oxotremorine to sham-operated rats is partially reduced in the adrenal medulla by muscarinic blockade, and completely in the cortex. This effect of methylatropine in regard to cortical ODC activity was not apparent in the other experiments with intact or unilaterally splanchnicotomized (unoperated side) rats. The results with unilaterally splanchnicotomized rats and those with transected spinal cord suggest that oxotremorine-induced modifications of adrenal ODC activity are centrally mediated, above the level of origin of the splanchnic nerves in the spinal cord (T_8–10). Experiments with hypophysectomized rats show that the response of the adrenal cortex to oxotremorine is entirely mediated by the hypophysis.     

Cell wall composition of the carrageenophyte Kappaphycus alvarezii (Gigartinales, Rhodophyta) partitioned by wet sieving

The cortical and medullary cells of Kappaphycus alvarezii fractions were screened by wet sieving after aqueous extraction of carrageenans. The cell populations obtained showed a clear partition between these two cell types. The main monosaccharide in hydro-insoluble cell walls was cellulosic glucose (70% dry weight), the crystallinity of which was shown torange from 20% in the cortical cells to 45% in the large medullary cells (over 250 μm diameter). Minor monosaccharides in the insoluble fraction were galactose, 3,6-anhydrogalactose (indicating presence of residual carrageenans), mannose and xylose. However, the major part of the remaining galactose probably originated from another galactoglycan strongly linked to insoluble polymers in the large medullarycell walls. The mannose concentration was maximum in the cortical cells and decreased with increasing size of the medullary cells. Thus, besides cellulose, two other types of polysaccharides were detected in insolublecell walls, mannoglycans and galactoglycans in cortical and medullary cellwalls, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Embryologic development of rat adrenal medulla in transplants to the anterior chamber of the eye

The morphological development and plasticity of embryonic and postnatal rat adrenal medullary cells were studied in homologous adrenal grafts to the anterior chamber of the eye. The eyes of recipient rats were adrenergically denervated 10 days prior to grafting by extirpation of the superior cervical ganglion in order to increase levels of NGF and NGF-like activities in the iris. Grafts taken at the 15th day of embryonic development (E15), i.e., at the beginning of immigration of medullary progenitor cells into the adrenal cortical anlagen, contained no cortical or mature medullary cells after 2 weeks in oculo. Numerous sympathoblastic cells, however, were located at the anterior surface of the iris. E 16 and E 17 transplants showed abundant mature cortical tissue after 2 weeks. Small groups of medullary cells with the ultrastructural characteristics of mature pheochromoblasts or young chromaffin cells were interspersed among cortical cells without forming a discrete medulla. Neuronal cells were exclusively found outside the cortical cell mass. Sympathoblasts grew at the surface of the iris, while young sympathetic nerve cells, which were invested by Schwann cells and received synaptic axon terminals, were embedded into the stroma of the iris. Grafting of E 21 adrenals yielded very similar results except that, in a few instances, young chromaffin cells were located outside the cortex and sympathetic nerve cells were seen to be in close contact with cortical cells. In transplants of adult medullary cells typical mature adrenaline and noradrenaline cells were clearly distinguishable after 8 weeks even in the absence of cortical cells. The only indication of phenotypical changes in these cells was the formation by some of them, of neuritic processes which could be visualized in glyoxylic acid-treated whole mounts of irises. These results are compatible with the idea that embryonic adrenal medullary cells have the environmentally controlled potential to develop along the neuronal or endocrine line, but could also be interpreted in terms of a selection of a specific subpopulation with predetermined potentialities by a specific microenvironment. Moreover, these results suggest that increasing differentiation of medullary cells is accompanied by progressive restrictions in their genetic program, which eventually prevent full transdifferentiation of mature chromaffin into neuronal cells.     

p60c-src activity detected in the chromaffin granule membrane

Using monoclonal antibodies specific for p60c-src we have detected high levels of this kinase in adrenal medullary chromaffin tissue and in highly purified chromaffin granule (secretory vesicle) membranes. An immune complex kinase assay was applied to fractions of adrenal medullary tissue resolved on sucrose density gradients. Thirty-seven per cent of the total tissue p60c-src activity was found in association with chromaffin granule or granule membrane markers. Localization of a significant fraction of total cellular p60c-src activity to this secretory vesicle membrane suggests that the kinase may function in the regulation of neurotransmitter release.     

An enzymecytochemical study on the adrenal of the freshwater teleost, Clarias batrachus (L.).

The adrenal homologue of C. batrachus is distributed around the postcardinal vein in the pronephric head kidney. The cortical cells are round or oval in shape. They showed positive reaction for total lipid, glycogen and ascorbic acid. Their intense delta5-3beta HSDH activity indicates their capacity for steroid biosynthesis. In addition, the cortical cells of C. batrachus exhibited strong G-6-PD, NADPH diaphorase, NADH diaphorase, MAO and weak SDH and LDH activity. The presence of MAO suggests the aminergic control of the adrenal in this species and the silver positive fibres seen the cortical cells were hypertrophied, degranulated and the lipid content was also decreased. The chromaffin or medullary cells were distributed in groups among the cortical cells. They are largely oval or angular in shape. They react positively to ferric ferricyanide, chromaffin and argentaffin reactions and ascorbic acid test.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Stimulating effect of cocaine in rats with different levels of blood esterase activity

It was shown in experiments on male random-bred rats that motor stimulating effect of cocaine (10 mg/kg, intraperitoneally) was dependent on the spectrum of blood serum esterase activity. The determination of esterase spectrum was performed by disc-electrophoresis in 7.5% PAAG (50-75 gamma of protein per gel), with enzyme activity estimated using alpha-naphthylacetate. Proserine (10(-5) M) and butyrylthiocholine controls were used. Motor stimulating effect of cocaine in animals with 3 esterase fractions was 3 times higher than in animals with 4 enzyme fractions.     

Histochemical evidence of three types of esterases in the adrenal medulla of the rat

Summary Esterases of the adrenal medulla have been studied histochemically using alpha-naphthyl acetate and butyrate as substrates, Blue RR Salt as a coupler and eserine and E, 600 as inhibitors. Three types of esterase activity were thus demonstrated: (1) cholinesterase activity in the nerve fibres, ganglion cells and secretory medullary cells; (2) eserine resistant but E. 600 sensitive esterase activity in the ganglion cells and secretory cells; (3) E. 600 resistant activity in strongly positive, unidentified cells scattered in the medulla. The histochemical picture was essentially similar in sections of formalin-fixed tissue and in fresh sections subjected to the voltage gradient employed for electrophoretic separation of esterases. It is concluded that esterases histochemically demonstrable in sections are desmo-enzymes and at least to a major part different from the lyo-enzymes which can be separated by starch gel electrophoresis.With 6 Figures in the Text     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

Characteristics of acid esterase in Wolman's disease

The characteristics of acid esterase from the patient with Wolman's disease, a rare familial lipidosis, were studied. Enzymatic analysis as well as mineral analysis were performed on the patient's liver, spleen, and adrenal glands. Acid esterase was low in the patient's leucocytes and other affected tissues. Further enzymatic study with subcellular fractions of the liver in both patient and control subject revealed that acid esterase was mostly localized in the membrane of lysosomes. The lysosomal esterase was unaffected by Ca2+, Mg2+, EDTA, E600 (microsomal esterase inhibitor), and it was less inhibited by NaCl than other fractions. Studies with those inhibitors showed that acid esterase has different properties compared to other lipases, such as lipoprotein lipase, adipose tissue lipase, and hepatic microsomal lipase. Studies with inhibitors also gave a negative view on a possible suppressive interaction of the high content of calcium in the target organs with acid esterase in Wolman's disease.     

The periodic acid-schiff reaction in the adrenal medulla

Summary The PAS reaction in the adrenal medulla of rat, rabbit, hamster, ox, pig and sheep was investigated. The medullary cells were positive in cryostat sections and potassium dichromate fixed material but not in formaldehyde fixed paraffin sections. The latter result is due mostly to the extraction of PAS positive lipids and loss of PAS positive proteins. No glycogen was detected in the chromaffin cells histochemically. The catechol amines played no part in the PAS reaction unless the fixative contained dichromate. The connective tissue elements were also PAS positive, and the nerve fibres in ox, sheep and pig. Periodate cannot be used to differentiate between adrenaline and noradrenaline storing cells.     

Mixed Corticomedullary Carcinoma of the Adrenal Gland: A Case Report

ObjectiveTo report the case of a 78-year-old woman with mixed corticomedullary carcinoma of the adrenal gland, and to review other reported lesions that exhibit clinical and/or histopathologic features of both adrenal cortical and medullary differentiation.MethodsWe describe the patient’s clinical findings and laboratory test results, as well as the gross and histopathologic features of her tumor. We also review the literature pertaining to mixed corticomedullary adenomas and cortical tumors with clinical features of pheochromocytoma, and vice versa.ResultsA 78-year-old woman with a 10-cm left adrenal mass was hospitalized for management of hypertensiveurgency. Laboratory workup revealed elevated urinary metanephrine excretion and elevated serum dehydroepiandrosterone sulfate levels. She underwent left adrenalectomy. Pathologic examination of the lesion showed mixed cortical and medullary histologic characteristics, as well as gross and microscopic evidence of malignancy. Including the present case, we identified 17 cases of neoplasms that exhibit features of mixed corticomedullary differentiation.ConclusionsThis report represents the first documented case of mixed corticomedullary carcinoma. Several benign lesions combine clinical, biochemical, and/or histopathologic evidence of both adrenal cortical and medullary differentiation, including mixed corticomedullary adenomas and corticotropin-secreting pheochromocytomas. The differential diagnosis of a lesion with mixed cortical and medullary features should also include a malignant neoplasm. (Endocr Pract. 2012;18:e37-e42)     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship.

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58-69% recovered in the 100 000 X g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 +/- 13.6% (S.D.) and triacylglycerol lipase 11.9 +/- 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, The hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent Km for substrate and molecular weight 3-10(5)-6-10(5) by gel filtration and a minor component of lower apparent Km and heterogeneous molecular weight above 1 million, which was found mostly in complex and lipid.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.