The response of rat adrenal medulla to oxytocin

Effects of oxytocin (OT) on the adrenal chromaffin tissue of male rats were examined by coupled morphometric and biochemical techniques. Synthetic OT was administered in doses of 0.14 and 0.25 IU/100 g/d during 7 or 10 consecutive days and the effects were followed 1, 24, 72 and 168 hours after the last injection. The function and structure of chromaffin cells were affected by the higher dose of OT only. They caused divergent responses on their amine contents. Adrenaline, noradrenaline and dopamine contents were increased, while serotonin content was decreased. These changes were different in duration and time of incidence. Stereological analysis showed an enhanced number of chromaffin cells and an increase in their total volume. The parallelism between the changes in chromaffin cell number and the catecholamine content strongly suggests a mitogenic effect of the applied OT.     

The acetylcholine receptor of the adrenal medulla.

Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [¹²⁵1]α-bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α-bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d-tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α-bungarotoxin. Perfusion experiments showed the acetylcholine receptor to be entirely nicotinic. Stimulation by nicotine was inhibited by atropine and decamethonium, as well as by hexamethonium. Calculated dissociation constants for these antagonist-receptor interactions were in the range of 1 to 3 × 10^?5 m. α-Bungarotoxin failed to inhibit nicotine-stimulated catecholamine release in the perfused adrenal, most likely because of its limited diffusion into the gland.     

Characterization of the rat adrenal medulla cultured in vitro

Summary A wide variety of experimental animal models have been used to investigate the mechanisms of synthesis, storage, and release of catecholamines. Whereas in vivo experimental models are situated at one end of the spectrum, cell culture models are situated at the other end. In the present study, we have characterized various aspects of the rat adrenal medulla cultured in vitro as a whole tissue, aiming to establish a new experimental model in between in vivo animal models and cell culture models. We adapted a bottle rotator system commonly used for culturing rodent whole embryos. Changes in histology, activities and mRNA levels of catecholamine-synthesizing enzymes, and concentrations of catecholamines in the adrenal medulla were studied. In addition, the effects of cholinergic stimulation on catecholamine release from the adrenal medulla were examined. Overall the results indicate that various aspects of the adrenal medulla become stable after 4 d of culture and the adrenal medulla at this stage releases catecholamines in response to cholinergic stimulation. The whole adrenal medulla culture system may be a useful tool for investigating catecholamine-related functions dependent on intercellular reactions or communications.     

Tissue-type plasminogen activator in rat adrenal medulla

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

High-affinity angiotensin receptors in rat adrenal medulla

Angiotensin II receptors have been quantitated in single rat adrenal medullas by incubation of tissue sections with 125I-[Sar1]-AII, autoradiography with exposure to 3H-sensitive Ultrofilm, computerized densitometry and comparison with 125I-labelled standards. Rat adrenal medulla contains a single class of high affinity AII receptors with a Ka of 0.84 +/- 0.02 X 10(9) M-1 and a Bmax of 3259 +/- 502 fmol/mg protein, one of the highest densities in AII receptors found in rat tissues. These observations provide evidence for a local site of action of AII in the release of adrenal medullary catecholamines.     

Tissue-type plasminogen activator in rat adrenal medulla

Summary Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenalin. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Changes in the adrenal medulla of the rat during immobilization stress

A combined histofluorescent, light optic and electron microscopic investigation has been performed to study the medulla of the adrenals in intact rats (August strain) and immediately after immobilization for 30 h and 24 h after. In the intact animals heteromorphism of the medulla is demonstrated; this depends on the fact that adrenocytes are at different stages of the secretory cycle. Immobilization for 30 h results in synchronization of secretion; this is determined by adaptation of the adrenal system to immobilization. One day after immobilization restoration of heteromorphism in chromaffinocytes is demonstrated. The changes described are compensatory-adaptive reactions of the adrenal medulla to the stress in the animals survived.     

Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.     

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.     

Growth characteristics of postnatal rat adrenal medulla in culture

Explants and enzyme-dispersed cells of adrenal medulla from 10-12 day old rats were studied in culture for up to 3 weeks. Adrenomedullary chromaffin cells, nerve cells and satellite cells were clearly discernible. The nerve cells were few in number and did not show catecholaminespecific fluorescence. Chromaffin cells stored catecholamines, as judged by the Falck and Hillarp method, in varying amounts decreasing with age of the cultures and the distance from the explants. Exocytosis profiles observed with the electron microscope suggested that cultured chromaffin cells also released catecholamines. Moreover, the cells formed processes and frequently migrated into the outgrowth. After 6 days in culture, the great majority of chromaffin cells stored noradrenaline as revealed by electron microscopy with few adrenaline-storing cells being visible. Granular vesicles (approximately 80-240 nm in diameter) with cores of different electron densities were occasionally present in the same cell suggesting the occurrence of mixtures of primary and secondary amines. Apart from "chromaffin" granules, small clear and dense-cored vesicles (approximately 40-60 nm) were found both in the somata and cell processes. Chromaffin cells and their processes were often closely apposed and occasionally formed specialized attachment zones. As a whole, chromaffin cells in culture resembled small granule-containing cells in sympathetic ganglia. 0.5 mM dbcAMP prevented dedifferentiation of chromaffin cells as judged by the lack of processes, the size and amount of "chromaffin" granules and the high number of adrenaline-storing cells present after 6 days in culture. NGF caused a striking increase in the number of axons growing out from explants.     

PhenylethanolamineN-methyltransferase from the brain and adrenal medulla of the rat

Two different molecular forms of phenylethanolamineN-methyltransferase (PNMT, EC 2.1.1) have been isolated from the brain and adrenal glands of the rat as indicated by certain of their physicochemical properties, such as: molecular weight estimated on the basis of gel chromatography of Sephadex G-100; pH optima; electrophoretic mobility on acrylamide gel; and steady-state kinetic parameters. The highK _m value for the brain PNMT has been assumed to be responsible for the low methylation ratio between norepinephrine and epinephrine in the CNS.     

Dynorphin and enkephalins in adrenal paraneurones. Opiates in the adrenal medulla

Immunoreactive dynorphin (ir-Dyn), immunoreactive leucine-enkephalin (ir-Leu-Enk) and various other neuropeptides were measured in acid extracts of bovine adrenal medulla and isolated adrenal chromaffin cells. Their respective levels ranged as follows: Leu-Enk greater than Dyn greater than bombesin greater than vasoactive intestinal peptide (VIP) greater than neurotensin greater than substance P. Comparisons of the total catecholamine levels with the levels of Leu-Enk in both extracts gave ratios in the same order of magnitude (2600, tissue extract and 5000, cell extract). However, the catecholamine/Dyn ratio in the tissue extract (138 000) was much higher than that found in the cell extract (20 180), suggesting a possible selective degradation of Dyn in tissue extract as compared with cell extract or an induction of Dyn biosynthesis in cells which have been isolated from their natural microenvironment. Immunofluorescence staining of isolated chromaffin cell sections revealed the presence of ir-Dyn in 5 to 10% of the total cell population. To localize ir-Dyn in regard to Leu-Enk and catecholamines, adrenal chromaffin cells were separated into three populations (I, II, and III) on a stepwise bovine serum albumin (BSA) gradient. Relative high levels of ir-Dyn were measured in cell layer I (4 pmol/10(6) cells), a cell population enriched in noradrenaline. However, ir-Leu-Enk was more concentrated in cell layers II and III (5.3 and 8.3 pmol/10(6) cells), two populations enriched in adrenaline. Isolation and high pressure liquid chromatography (HPLC) analysis of adrenomedullary Dyn indicated the presence of at least five molecular forms corresponding to Dyn-(1-11), Dyn-(1-12), Dyn-(1-13), Ala-containing-Dyn-(1-13) and a nonidentified molecule eluting closely to Dyn-(1-13). These data indicate that adrenal ir-Dyn and ir-Leu-Enk have distinct cellular distributions. In addition, the identification of Dyn fragments in bovine adrenal medulla indicates that these short peptides may be considered as natural active forms of Dyn.     

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla.

Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections. Four types of primary catecholamine-storing cells were identified. NA1 cells contain cytoplasmic granules up to 0.3 mum in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA2 cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA3 cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA4 cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence. The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA1 cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA2 cells correspond to this latter population (175 nm). NA3 cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA4 cells being in the dimension of 80 nm. Granules in NA1 and NA2 cells show uniformly high density, whereas those in NA3 and NA4 cells display cores of varying density. Granules with moderately dense cores in NA3 and NA4 cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.     

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.