Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.     

Upregulation of neuronal nitric oxide synthase mRNA and protein in adrenal medulla of water-deprived rats.

Experiments were performed to investigate whether adrenal neuronal nitric oxide synthase (nNOS) mRNA and protein expression are responsive to alterations in body volume. Using an RT-PCR technique, the relative quantities of nNOS mRNA as well as the tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA in the adrenals of water-deprived rats significantly increased from 12 hr to 4 days. In situ hybridization and immunohistochemical study showed that water deprivation activated nNOS mRNA and protein expression in the adrenal medulla. Four days after water deprivation, nNOS protein expression determined by Western blot significantly increased in the adrenal gland. Our results are the first to demonstrate that nNOS syntheses in the adrenal medulla are markedly increased in water-deprived rats. This study also indicates that the upregulation of nNOS synthesis of the adrenal medulla is associated with the activation of adrenal medullary function in the face of volume depletion.     

Immunohistochemical identification and comparison of glial cell lineage in foetal, neonatal, adult and neoplastic human adrenal medulla

The differentiation of glial cells in developing, neonatal, adult and neoplastic human adrenal medulla has been studied immunohistochemically. From 8 to 28 weeks' gestational age, S-100 protein and its β-subunit revealed two different glial cell populations in adrenal glands, namely Schwann-like and sustentacular cells. Schwann-like cells were spindle-shaped cells forming a continuous layer around groups of sympathetic neuroblasts, often in contact with Schwann cells of nerve fibres entering neuroblastic groups. Sustentacular cells were round or oval cells with dendritic cytoplasmic processes; they were not associated with nerve fibres and mingled both with sympathetic neuroblasts and differentiating chromaffin cells. The developmental fate of Schwann-like cells was different from that of sustentacular cells. Schwann-like cells disappeared from the 28th week of gestational age, in association with the disappearance of sympathetic neuroblastic groups, and they were rarely found in neonatal and adult adrenal medulla. In contrast, sustentacular cells persisted between medullary chromaffin cells, and their number and dendritic cytoplasmic processes progressively increased from foetus to adult. In eight cases of primitive adrenal neuroblastic tumours of neonatal age (five undifferentiated neuroblastomas and three ganglioneuroblastomas), Schwann-like cells were found at the periphery of tumoral nests with a lobular growth pattern, while rare sustentacular cells were associated with neuroblasts. In two cases of adult phaeochromocytomas, only sustentacular cells were detected between chromaffin tumoral cells. Our findings suggest that the glial cell types and their distribution in primitive adrenal medulla tumours closely resemble those observed during development in the groups of adrenal sympathetic neuroblasts and in the clusters of chromaffin cells     

Das unterschiedliche Bild organspezifischer Strukturen im Glomus caroticum und Nebennierenmark

Zusammenfassung Die organspezifischen Strukturen im Glomus caroticum und Nebennierenmark zeigen unter verschiedenen experimentellen Bedingungen ein unterschiedliches Bild. So läßt sich unter anderem durch Modifizierung der Fixationsmedien (Glutaraldehyd, Osmiumsäure in Kombination mit verschiedenen Puffern) eine optimale Differenzierung der zellulären Strukturelemente von Glomuszellen erreichen. An den osmiophilen Vesikeln fällt eine starke Variabilität ihres Erscheinungsbildes auf. Feinstrukturelle Unterschiede werden in Elektronendichte des Vesikelinhaltes, Größe und Ausbildung des Hofes sowie in Aufbau und Verlauf der Membran deutlich. Auf Grund dieser Kriterien lassen sich im wesentlichen 4 Haupttypen der Granula von seltener vorkommenden Sonderformen abgrenzen.

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Ultrastructure of secretory granules and other specific inclusions in the carotid body and adrenal medulla

Summary The organospecific structures of the carotid body and the adrenal medulla show a different appearance under various experimental conditions. Thus, it is possible to obtain an optimal differentiation of the cellular structure elements of the carotid body cells by modifying the fixatives (glutaraldehyde, osmic acid, in combination with various buffer systems).Especially the osmiophilic vesicles show a great variability in their appearance. Ultrastructural differences become evident in the electron-density of the vesicle content, in the size of the electron-lucent space (halo of the dense core), as well as in the structure and conduct of the membrane. Based on these criteria four main types of granules can be said to occur in the carotid body. These can be distinguished from more uncommon special types.

Für technische Hilfe danken wir Frl. B. Ferdinand und Frl. B. Schmaloer.     

Immunohistochemical identification and comparison of glial cell lineage in foetal, neonatal, adult and neoplastic human adrenal medulla

The differentiation of glial cells in developing, neonatal, adult and neoplastic human adrenal medulla has been studied immunohistochemically. From 8 to 28 weeks' gestational age, S-100 protein and its β-subunit revealed two different glial cell populations in adrenal glands, namely Schwann-like and sustentacular cells. Schwann-like cells were spindle-shaped cells forming a continuous layer around groups of sympathetic neuroblasts, often in contact with Schwann cells of nerve fibres entering neuroblastic groups. Sustentacular cells were round or oval cells with dendritic cytoplasmic processes; they were not associated with nerve fibres and mingled both with sympathetic neuroblasts and differentiating chromaffin cells. The developmental fate of Schwann-like cells was different from that of sustentacular cells. Schwann-like cells disappeared from the 28th week of gestational age, in association with the disappearance of sympathetic neuroblastic groups, and they were rarely found in neonatal and adult adrenal medulla. In contrast, sustentacular cells persisted between medullary chromaffin cells, and their number and dendritic cytoplasmic processes progressively increased from foetus to adult. In eight cases of primitive adrenal neuroblastic tumours of neonatal age (five undifferentiated neuroblastomas and three ganglioneuroblastomas), Schwann-like cells were found at the periphery of tumoral nests with a lobular growth pattern, while rare sustentacular cells were associated with neuroblasts. In two cases of adult phaeochromocytomas, only sustentacular cells were detected between chromaffin tumoral cells. Our findings suggest that the glial cell types and their distribution in primitive adrenal medulla tumours closely resemble those observed during development in the groups of adrenal sympathetic neuroblasts and in the clusters of chromaffin cells     

Proenkephalin: A general pathway for enkephalin biosynthesis in animal tissues

Guinea pig adrenal, brain, and myenteric plexus have been shown to contain many polypeptides that yield free enkephalins on digestion with trypsin and carboxypeptidase B. The enkephalin-containing polypeptides (ECPs) range from 500 to >20,000 daltons and show similarities in their chromatographic behavior to the ECPs present in the chromaffin granules of the bovine adrenal medulla. Furthermore, the heptapeptide [Met]enkephalin-Arg⁶-Phe⁷, that is now known to represent the carboxyl terminal sequence of the proenkephalin found in bovine adrenal medulla (Gübler et al. (1982) Nature (London), in press), was identified in all three guinea pig tissues. It appears that processing of a proenkephalin similar to the one in adrenal medulla represents a general pathway for enkephalin biosynthesis in animal tissues.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

CONSTANCY OF DNA CONTENT IN ADRENAL MEDULLA NUCLEI OF COLD-TREATED RATS

Reports of changes in DNA content of certain types of cells following exposure to conditions of stress has led to the suggestion that two kinds of DNA may be present. One is genetic DNA, and the other is called "metabolic" DNA. In a further attempt to investigate the possibility of this phenomenon, determinations of DNA content were made on Feulgen-stained nuclei of adrenal glands and kidneys in cold-treated rats. Feulgen-stained nuclei were measured by two-wavelength microspectrophotometry. Particular attention was given to the handling of the smears in hydrolysis and staining. Mean values of Feulgen-DNA contents in a total of 720 nuclei demonstrated (a) a constancy of DNA content within 2% in individual nuclei both in adrenal medulla and kidney cortex, (b) no more than an average of 2% difference in DNA content between control and experimental nuclei, and (c) no more than an average of 1.5% difference in DNA content between normal kidney cortex nuclei and normal adrenal medulla nuclei. These results confirm the view that the more precise the measurement, the more accurately the constancy rule is obeyed. Moreover, there is no support for the concept of a metabolic DNA in the rat adrenal medulla.     

Characterisation of Met-enkephalin(Arg6,Phe7) immunoreactivity in human adrenal and human phaeochromocytoma

The molecular forms of opioid peptides in human adrenal have not been well characterised. These peptides are predominantly derived from the proenkephalin A precursor, which has the sequence of Met-enkephalin(Arg⁶,Phe⁷) as its carboxyl terminus. We have looked in the present study at the subcellular distribution and the molecular form of immunoreactivity to this sequence in post-mortem human adrenal medulla and in phaeochromocytoma. In the human adrenal homogenates, the immunoreactivity distributes on a sucrose gradient in a manner consistent with localisation in chromaffin granules. On chromatography, the immunoreactivity from adrenal medulla is predominantly in the heptapeptide form; the intermediate (3000–4000) molecular weight material is only a minor component of immunoreactivity, in contrast to bovine tissue extracts where this is the major form of immunoreactivity. In the phaeochromocytoma extracts, the heptapeptide sequence again predominates over a minor amount of intermediate sized material. The results are discussed in terms of post-mortem changes, precursor processing and the function of the adrenal medulla.     

Neurohypophysial-adrenal gland responses to water-deprivation in the rose-ringed parakeet.

The aim of the present investigation is to study the relative influence of neuohypophysis, adrenal cortex and adrenal medulla under dehydration stress in the parakeet. Birds subjected to dehydration for 7 days lost body weight. Neurosecretory material (NSM) was partially depleted from the neurohypophysis. Adrenal gland weight was increased followed by a hypertrophy of the cortical tissue. A fall in adrenal cholesterol and ascorbic acid level was marked. Adrenaline and noradrenaline contents of the adrenal medulla were suppressed as was evident from cytochemical and biochemical findings. It is suggested that neurohypophysis, adrenal cortex and adrenal medulla are involved in maintaining water homeostasis in this avian species.     

白暨豚肾上腺的研究

白鱀豚肾上腺重与体重的平均比值为0.25克/公斤,皮质体积与髓质体积的比值为6.59。白鱀豚肾上腺的组织结构与其它海豚相似,它有比较发达的球状带。讨论了白鱀豚肾上腺形态变化在年龄生长、授乳等生理过程和在自然环境、豢养环境生态适应上的意义。并报道了一例罕见的白鱀豚肾上腺先天性表皮样囊肿。       

Morphometric and histological studies on the adrenal glands of the camel Camelus dromedarius

The adrenal gland of the camel consists of an outer cortex and an inner medulla. The general disposition of the cortex and medulla, however, differs occasionally from that of other mammals. Extensions of medulla could reach as far as the periphery of the cortex. Islet of medullary tissue may be found in sections of the cortex and cortical tissue consisting of all zones of the cortex may occur around arteries or nerves in the medulla. The medulla may be separated from the cortex by connective tissue especially in old camels. The arrangement of noradrenaline-secreting cells is different from that in other ruminants; they are found in groups scattered between the adrenaline-secreting cells. Bundles of smooth muscle occur in venules at the corticomedullary interface. Accessory adrenal glands are found embedded in the renal fat. They are similar in structure to the adrenal gland. The adrenal cortex forms 74% of the volume of the gland and the ratio of the cortex to medulla is 4:1. The zona glomerulosa, fasciculata and reticularis constitute about 13%, 53%, and 29% by volume of the cortex, respectively.     

The effects of enalapril maleate and cold stress exposure on tyrosine hydroxylase activity in some rat tissues

Enalapril is a highly specific and competitive inhibitor of angiotensin-I converting enzyme (ACE) and thus belongs to the category of ACE inhibitors. The beneficial effects of ACE inhibitors appear to result primarily from the suppression of the plasma renin-angiotensin-aldesterone system. This study was designed to detect the effects of enalapril maleate and cold stress on tyrosine hydroxylase (TH) activity in adrenal medulla, heart and hypothalamus in rat. In cold stress treatment (exposed to 8 degrees C cold for 48 h) TH activity was found to be raised significantly (p < 0.05) in adrenal medulla, hypothalamus and heart tissues. In the adrenal medulla, hypothalamus and heart tissues, TH activity of enalapril maleate treated rats (10 mg kg(-1) body weight) group was not raised significantly (p > 0.05). Following intraperitoneal injection of enalapril maleate (10 mg kg(-1) body weight) the rats were exposed to 8 degrees C cold for 48 h. After cold stress and enalapril maleate treatment no statistically significant change in tyrosine hydroxylase activity was detected in adrenal medulla, hypothalamus or heart (p > 0.05). The results of our studies show that enalapril maleate blocks the effect of cold stress on the regulation of TH activity.     

Disseminated malignant phaeochromocytoma: localisation with iodine-131-labelled meta-iodobenzylguanidine.

Meta-iodobenzylguanidine, a guanethidine analogue, is a newly synthesised substance capable of imaging the adrenal medulla. In a woman in whom phaeochromocytoma has been diagnosed iodine-131-labelled metaiodobenzylguanidine was given intravenously; gamma-camera images showed bilateral adrenal tumours and uptake corresponding to bone and liver metastases. 131I-meta-iodobenzylguanidine is effective in localising phaeochromocytomas, and the technique is safe, specific, and non-invasive.     

Changes in the catecholamine metabolism in the adrenal medulla of male hamsters with experimental hyperprolactinemia

1. Prolactin (PRL) can play a role as a physiological modulator of adrenal medulla function in several rodents. 2. We have examined the effects of hyperprolactinemia induced by ectopic pituitary grafts in Syrian hamsters on the adrenal medulla contents of catecholamines (CA) and their metabolites, as well as on the activities of several enzymes involved in the metabolism of these amines. 3. Increases in the peripheral levels of PRL in these animals were associated with decreases in adrenal medulla weight and increases in adrenal medulla contents of norepinephrine, epinephrine and vanilmandelic acid, the main degradative metabolite of CA, while adrenal medulla contents of the O-methylated derivatives of CA, normetanephrine and metanephrine, were unaltered. 4. These changes were correlated with increases in the adrenal medulla activity of monoamine oxidase, while the activities of tyrosine hydroxylase, phenylethanolamine-N-methyl transferase and catechol-O-methyl transferase were unaltered. 5. These results indicate that PRL is able to act on the adrenal medulla of hamsters by increasing the ability of these cells to metabolize CA via oxidative deamination.     

Cytokine Interactions with Adrenal Medullary Chromaffin Cells

It is generally accepted that a bi-directional or reciprocal interaction occurs between the immune and neuroendocrine systems, and that this relationship is important for the appropriate physiological functioning of both systems. Similarly, an imbalance in this relationship may contribute to a number of pathologies, most notably those relating to stress. The aim of this article is to consider the interaction of cytokines with the adrenal medulla, a potentially important player in this relationship. The chromaffin cells of the adrenal medulla release catecholamines and a range of biologically active peptides in response to a wide variety of stress-related signals. A growing body of evidence indicates that this stress response is influenced by, and in turn has influence upon, immune signalling. This brief review will focus primarily on the best-described adrenal medullary active cytokines, namely interferon-α, interleukin-6, interleukin-1α/β and tumour necrosis factor-α. In each case, three key issues will be addressed: the physiologically relevant source of the cytokine; the intracellular signalling events arising from activation of its receptor and finally the cellular consequences of such activation in terms of modulation of gene expression and the secretory output of the chromaffin cells.     

Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice.

Deficiency of 21-hydroxylase (21-OH), one of the most common genetic defects in humans, causes low glucocorticoid and mineralocorticoid production by the adrenal cortex, but the effect of this disorder on the adrenomedullary system is unknown. Therefore, we analyzed the development, structure, and function of the adrenal medulla in 21-OH-deficient mice, an animal model resembling human congenital adrenal hyperplasia. Chromaffin cells of 21-OH-deficient mice exhibited ultrastructural features of neuronal transdifferentiation with reduced granules, increased rough endoplasmic reticulum and small neurite outgrowth. Migration of chromaffin cells in the adrenal to form a central medulla was impaired. Expression of phenylethanolamine-N-methyltransferase (PNMT) was reduced to 27 +/- 9% (P<0.05), as determined by quantitative TaqMan polymerase chain reaction, and there was a significant reduction of cells staining positive for PNMT in the adrenal medulla of the 21-OH-deficient mice. Adrenal contents of epinephrine were decreased to 30 +/- 2% (P<0. 01) whereas norepinephrine and dopamine levels were reduced to 57 +/- 4% (P<0.01) and 50 +/- 9% (P<0.05), respectively. 21-OH-deficient mice demonstrate severe adrenomedullary dysfunction, with alterations in chromaffin cell migration, development, structure, and catecholamine synthesis. This hitherto unrecognized mechanism may contribute to the frequent clinical, mental, and therapeutic problems encountered in humans with this genetic disease.     

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.     

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.     

Selective activation of the adrenal medulla during acute bilateral carotid occlusion and its modulation by alpha-adrenergic receptors in the rat

The sympathoadrenal activity was studied during baroreflex stimulation in chloralose anesthetized rats. Circulating norepinephrine (NE) and epinephrine (E) levels were used as indices of sympathetic fiber and adrenal medulla activities, respectively, under basal conditions and during a 1-min bilateral carotid occlusion (CO). In vagotomized rats, the CO induced a significant increase in mean arterial pressure (MAP) associated with an increase in circulating E levels, while this procedure did not alter blood pressure or circulating NE or E levels in intact animals. Following vagotomy, the baroreflex stimulation activated specifically the adrenal medulla, without alteration of the sympathetic fiber activity since the NE levels were not modified by the occlusion. Moreover, in support of that hypothesis, chemical sympathectomy did not decrease the pressure response to CO while bilateral adrenalectomy almost completely abolished this response. The elevation of circulating E induced by the CO was greatly potentiated by pretreatment with Yohimbine, a selective alpha 2-antagonist, and was completely abolished by administration of Clonidine, an alpha 2-agonist, while phenoxybenzamine, which is mainly an alpha 1-antagonist, did not potentiate significantly the E response to CO. These results therefore suggest that the baroreflex activation of the adrenal medulla induced by CO may be modulated in vivo via alpha 2-adrenergic receptors that could be localized on chromaffin cells.