Metabolic regulation of steroid steroidogenesis in isolated adrenal and adrenocortical carcinoma cells of rat. The incorporation of (20S)-20-(7- 3 H)hydroxycholesterol into deoxycorticosterone and corticosterone

Previous studies from these laboratories have demonstrated the following: (1) that cyclic 3′5′-AMP (c-AMP) phosphodiesterase activity of adrenocortical carcinoma 494 is only 20% of that found in the normal adrenal; (2) that corticosteroido-genesis in the isolated tumor cells is inhibited by ACTH, and (3) that the normal adrenal biosynthetic pathway from pregnenolone to corticosterone is intact but less active in the tumor. The present studies show that both the isolated adrenocortical carcinoma cell and the normal isolated adrenal cell of the rat have the capacity to transform (20S)-20-hydroxycholesterol into deoxycorticosterone and corticosterone. It is, therefore, proposed that the lack of stimulation by ACTH of corticosterone synthesis of the tumor cells cannot be explained by the absence of enzymes cleaving the cholesterol side chain. It is, therefore, postulated that a modified protein kinase may be present in the tumor which is not stimulated by c-AMP.     

Characterization of rat capsular adrenal (zona glomerulosa) (Na,K)-ATPase activity

A higher (Na,K)-ATPase activity was found in rat capsular (zona glomerulosa) than in decapsulated (zona fasciculata) adrenal. No unusual characteristics of this enzyme were found in the simulation with Na+ and K+ and the inhibition with ouabain. Angiotensin II, ACTH and serotonin, all potent stimulators of aldosterone biosynthesis, did not affect the enzyme. In conclusion, the characteristics of rat capsular adrenal (Na,K)-ATPase are identical to the classical enzyme. It is not the direct receptor or effector for stimulators of aldosterone biosynthesis but a supportive role in mediating the regulatory signal cannot be ruled out.     

Adaptation of the bank vole (Clethrionomys glareolus Schreber) to conditions of biogeochemical province with abnormally high content of nickel, cobalt and chromium

Morphophysiological characteristics and peculiarities of adrenal gland of bank vole (Clethrionomys glareolus) were studied in the area of natural biogeochemical province with abnormally high content of nickel, cobalt and chromium. The control population inhabited area with usual content of these elements. We used 4-factor analysis of variance to estimate the influence of geochemical conditions, phase of population cycle, sex and reproductive state on the morphophysiological characteristics of animals and functional activity of adrenal gland. Animals from area with high concentration of Ni, Co and Cr show an increase in relative mass of adrenal glands, fascicular zone of adrenal cortex, size of cells and their nuclei. All these changes can be considered as an evidence of increased secretion of glucocorticoids. It is shown that phase of population cycle influences fatness of animals, size of nuclei, cells and adrenal cortex. Females in comparison with males are characterized with higher indexes of liver and adrenal gland, as well as morphometric indexes of adrenal cortex. The maturation of animals is accompanied with increase in body mass, fatness and relative mass of adrenal glands, the size of cortex zone, nuclei and cells themselves. It is supposed that the effect of "geochemical factor" results in intensification of glucocorticoid secretion of adrenal costex, thus increasing non-specific resistance of animals inhabiting area with high concentration of heavy metals. Such factors as "phase of population cycle", "sex" and "reproductive state", influence mineralocorticoid activity, glucocorticoid and androgenic functions of adrenal cortex. Some factors show synergetic effect.     

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。       

IMMUNOLOGICAL STUDIES ON A MEMBRANE PROTEIN (CHROMOMEMBRIN B) OF CATECHOLAMINE-STORING VESICLES

Abstract— Rabbits were immunized with chromomembrin B, i.e. a membrane protein isolated from chromaffin granules of bovine adrenal medulla. When the rabbit sera were tested by immunodiffusion in the presence of various detergents, only negative results were obtained, whereas with complement fixation antibodies could be demonstrated. With this method the subcellular distribution of chromomembrin B in bovine adrenal medulla was determined. The results demonstrate that this protein is specifically localized in the membranes of chromaffin granules. In the mitochondrial and microsomal fractions it is present only in small amounts which are attributable to a contamination of these fractions with chromaffin granules. The subcellular distribution of chromomembrin R in bovine splenic nerves indicates that this antigen is also found in the membranes of noradrenalinestoring vesicles of sympathetic nerve. Chromomembrin B or a related antigen was detected in chromaffin grades isolated from pig and rat adrenal and in those isolated from a human phaeochromocytoma. It is also present in total membranes obtained from posterior and anterior hypophysis, but it is absent from membranes isolated from parotid gland, liver and adrenal cortex. This paper illustrates how a membrane protein which requires detergents for its solubilization can be characterized and measured by immunological methods.     

Trypanosoma cruzi: possible stimulatory factor(s) on brown adipose tissue of mice

Brown adipose tissue (BAT) was the most heavily infected tissue (mean 223 amastigotes/50 microscopic fields) by Trypanosoma cruzi in mice (P > 0.05) out of the 16 tissues examined. The second most infected group of tissues was the heart (mean 83 amastigotes), adrenal cortex (64), skeletal muscle (56), and pancreas (54). BAT and the adrenal cortex were only slightly infected in rats, and not infected at all in hamsters and guinea pigs.It appears that something is present in BAT, and in the adrenal cortex of mice that is physiologically attractive and growth stimulating to T. cruzi. Certain in vitro experiments with T. cruzi may be in order to determine whether certain steroid hormones may be stimulatory.     

Increased expression of (pro)renin receptor in aldosterone-producing adenomas

(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the present study is to clarify expression and possible pathophysiological roles of (P)RR in aldosterone-producing adenomas (APAs) and other adrenal tumors. Expression of (P)RR was studied by immunocytochemistry, western blot analysis and real-time RT-PCR in adrenal tumor tissues obtained at surgery. Immunocytochemistry showed that (P)RR was expressed in normal adrenal glands and tumor tissues of adrenocortical tumors including APAs. In the normal adrenal glands, positive (P)RR immunostaining was observed in both adrenal cortex and medulla, with higher (P)RR immunostaining observed in zona glomerulosa and zona reticularis. Positive (P)RR immunostaining was also observed in the adrenocortical tumors, with elevated (P)RR immunostaining found in APAs, particularly in compact cells. By contrast, no apparent (P)RR immunostaining was observed in pheochromocytomas. Western blot analysis showed a band of (P)RR protein in normal adrenal glands and adrenocortical tumors at the position of 35 kDa. The relative expression levels of (P)RR protein were higher in tumor tissues of APAs than in attached non-neoplastic adrenal tissues of APAs. Real-time RT-PCR showed that expression levels of (P)RR mRNA were significantly increased in tumor tissues of APAs compared with other adrenal tumor tissues and attached non-neoplastic adrenal tissues of APAs. The present study has shown for the first time that expression of (P)RR is elevated in tumor tissues of APAs, raising the possibility that (P)RR may play pathophysiological roles in APAs, such as aldosterone secretion and cell proliferation.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by double-staining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with Mr approximately 46,000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Summary Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by doublestaining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with M _r46000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

Role of PAC(1) receptor in adrenal catecholamine secretion induced by PACAP and VIP in vivo

The present study was conducted to investigate the functional implication of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC(1)) receptor in the adrenal catecholamine (CA) secretion induced by either PACAP-27 or vasoactive intestinal polypeptide (VIP) in anesthetized dogs. PACAP-27, VIP, and their respective antagonists were locally infused to the left adrenal gland via the left adrenolumbar artery. Plasma CA concentrations in adrenal venous and aortic blood were determined by means of a high-performance liquid chromatograph coupled with an electrochemical detector. Adrenal venous blood flow was measured by gravimetry. The administration of PACAP-27 (50 ng) resulted in a significant increase in adrenal CA output. VIP (5 microg) also increased the basal CA secretion to an extent comparable to that observed with PACAP-27. In the presence of PACAP partial sequence 6--27 [PACAP-(6--27); a PAC(1) receptor antagonist] at the doses of 7.5 and 15 microg, the CA response to PACAP-27 was attenuated by approximately 50 and approximately 95%, respectively. Although the CA secretagogue effect of VIP was blocked by approximately 85% in the presence of PACAP-(6--27) (15 microg), it remained unaffected by VIP partial sequence 10--28 [VIP-(10--28); a VIP receptor antagonist] at the dose of 15 microg. Furthermore, the CA response to PACAP-27 did not change in the presence of the same dose of VIP--(10--28). The results indicate that PACAP-(6--27) diminished, in a dose-dependent manner, the increase in adrenal CA secretion induced by PACAP-27. The results also indicate that the CA response to either PACAP-27 or VIP was selectively inhibited by PACAP-(6--27) but not by VIP-(10--28). It is concluded that PAC(1) receptor is primarily involved in the CA secretion induced by both PACAP-27 and VIP in the canine adrenal medulla in vivo.     

Immunocytochemical localization of metabotropic (mGluR2/3 and mGluR4a) and ionotropic (GluR2/3) glutamate receptors in adrenal medullary ganglion cells

The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.     

Structure of the adrenal glands of Greenland seal Pagophilus groenlandica Erxleben, 1777 (Phocidae)

The harp seal adrenal gland studied is alike that of terrestrial mammals, but has some peculiarities. Firstly, this is presence of the fetal cortex in the adrenal gland without any signs of involution and degeneration in adult animals. This phenomenon has been previously described only for the horse adrenal gland. The glomerular zone of the cortex peculiar for the adrenals in the terrestrial mammals in the species studied has the form of arches. This property is also characteristic only to the horse gland. The absence of intermediate and juxtamedullary zones also differs the harp seal adrenal gland from that of terrestrial mammals.     

Adrenal myelolipoma in the cottontop tamarin (Saguinus o. oedipus)

Two cases of myelolipoma in the cottontop tamarin (Saguinus o. oedipus) were found unequivocally within the adrenal gland, supporting an earlier suggestion that the adrenal gland was the possible origin of a juxtarenal myelolipoma in the same species. In the present study a 30-mm-diameter myelolipoma was present in the adrenal gland in case 1, whereas in case 2 the myelolipoma was only detected on histological examination. In case 1, changes in the adrenal gland were considered to be of minor clinical significance, whereas in case 2 the myelolipoma was an incidental finding.     

Binding sites for atrial natriuretic factor (ANF) in kidneys and adrenal glands of spontaneously hypertensive (SHR) rats

Binding sites for atrial natriuretic factor (ANF) were studied in kidneys and adrenal glands of 17 week old male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats by quantitative autoradiography using 125I-ANF-28. In kidney, 125I-ANF-28 binding sites were found in high concentrations in glomeruli and in much lower concentrations in the renal papilla. In adrenal gland, 125I-ANF-28 binding sites were highly localized to the zona glomerulosa and were of moderate density in the inner cortical regions. ANF binding sites did not occur in the adrenal medulla. The maximum binding capacity (Bmax) of 125I-ANF-28 was reduced by 50% in the kidney glomeruli of SHRs compared to WKY controls. In contrast, the affinity constant (Ka) for 125I-ANF-28 was elevated by 100% in kidney glomeruli of SHRs. There were no significant strain differences in values for Bmax or Ka for 125I-ANF-28 binding in the adrenal zona glomerulosa. These findings suggest that the natriuretic and diuretic actions of ANF within kidney glomeruli may be compromised in adult SHR rats and these alterations may contribute to the development and maintenance of hypertension in rats of this strain.     

Compensatory increase in adrenomedullary angiotensin-converting enzyme activity (kininase II) after unilateral adrenalectomy

Angiotensin-converting enzyme (ACE, kininase II, dipeptidyl carboxypeptidase, EC 3.4.15.1) was characterized in the adrenal medulla of male Sprague-Dawley rats. Rat adrenal medulla and lung ACE were similar in their susceptibility to Cl- activation and to the inhibition by EDTA, captopril, bacitracin and thiorphan, suggesting that rat adrenal medulla and lung ACE have similar properties. Changes in right adrenal weight and in adrenomedullary ACE activity 5 and 12 days following left unilateral adrenalectomy (UADX) were examined. Compensatory adrenocortical hypertrophy 12 days following UADX was associated with a significant increase in adrenal medullary ACE activity. This change was due not to a modified affinity of ACE for the substrate but to an alteration in ACE maximal velocity or number of available molecules. UADX had no effect on adrenocortical ACE activity. When UADX was combined with right splanchnic denervation, the increase in adrenomedullary ACE activity was blocked. The results support the existence of a functional ACE in adrenal medulla that is under neuronal control.     

In Adrenal Medulla Synaptophysin (Protein p38) Is Present in Chromaffin Granules and in a Special Vesicle Population

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.     

Co-localization of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON)

Neuropeptide tyrosine (NPY) is one of the most abundant and widespread peptides in the mammalian nervous system. Recent isolation and sequencing of the DNA encoding NPY has predicted the existence of a 97 amino acid precursor peptide. Proteolytic processing of this precursor could yield three separate peptide products, an N-terminal signal peptide, neuropeptide tyrosine and a 30 amino acid C-terminal flanking peptide (C-PON). Here, we present evidence that the predicted C-flanking peptide of NPY is widely distributed in both the central and peripheral nervous systems of several mammalian species including man, and has an identical distribution to NPY. It was also demonstrated, using correlative light microscopic immunostaining on serial sections and double electron microscopic immunocytochemistry, that C-PON and NPY immunoreactivities are co-localized in neuronal cell bodies of the brain cortex, sympathetic ganglion cells, norepinephrine-containing granules of the adrenal medulla and in human pheochromocytoma tumor cells.