Recruitment of governing elements for electron transfer in the nitric oxide synthase family.

At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O(2) was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.

Diversity-oriented synthesis of multisubstituted olefins through the sequential integration of palladium-catalyzed cross-coupling reactions. 2-pyridyldimethyl(vinyl)silane as a versatile platform for olefin synthesis.

A novel strategy for the diversity-oriented synthesis of multisubstituted olefins, where 2-pyridyldimethyl(vinyl)silane functions as a versatile platform for olefin synthesis, is described. The palladium-catalyzed Heck-type coupling of 2-pyridyldimethyl(vinyl)silanes with organic iodides took place in the presence of Pd2(dba)3/tri-2-furylphosphine catalyst to give beta-substituted vinylsilanes in excellent yields. The Heck-type coupling occurred even with alpha- and beta-substituted 2-pyridyldimethyl(vinyl)silanes. The one-pot double Heck coupling of 2-pyridyldimethyl(vinyl)silane took place with two different aryl iodides to afford beta,beta-diarylated vinylsilanes in good yields. The palladium-catalyzed Hiyama-type coupling of 2-pyridyldimethyl(vinyl)silane with organic halides took place in the presence of tetrabutylammonium fluoride to give di- and trisubstituted olefins in high yields. The sequential integration of Heck-type (or double Heck) coupling and Hiyama-type coupling produced the multisubstituted olefins in regioselective, stereoselective, and diversity-oriented fashions. Especially, the one-pot sequential Heck/Hiyama coupling reaction provides an extremely facile entry into a diverse range of stereodefined multisubstituted olefins. Mechanistic considerations of both Heck-type and Hiyama-type coupling reactions are also described.

Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy.

Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm. This is demonstrated with a rabbit liver P450 2B4. Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420. P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively. For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives. For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1). The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives. These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively. In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.

Roles of carbon monoxide in leukocyte and platelet dynamics in rat mesenteric during sevoflurane anesthesia.

BACKGROUND: Heme oxygenase 1 (HO-1), induced by a variety of stressors, provides endogenous carbon monoxide (CO) and bilirubin, both of which play consequential roles in organs. The current study aimed to examine whether induction of HO-1 and its by-products modulated endothelial interaction with circulating leukocytes and platelets evoked by sevoflurane anesthesia in vivo. METHODS: Rats, pretreated with or without hemin, were anesthetized with sevoflurane in 100% O2, and lungs were mechanically ventilated. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester and leukocyte behavior in mesenteric venules were visualized during sevoflurane anesthesia at 1,000 frames/s using intravital ultrahigh-speed intensified fluorescence videomicroscopy. To examine the mechanisms for the effects of HO-1 on leukocyte and platelet behavior, these studies were repeated with superfusion of either CO, bilirubin, or Nomega-nitro-L-arginine methyl ester (L-NAME). RESULTS: As reported previously, the elevation of sevoflurane concentration evoked adhesive responses of leukocytes, concurrent with platelet margination and rolling. Pretreatment with hemin, a HO-1 inducer, prevented such sevoflurane-elicited changes in the microvessels. These changes were restored by zinc protoporphyrin IX, a HO inhibitor, and repressed by CO but not by bilirubin. During sevoflurane anesthesia, however, nitric oxide suppression by L-NAME deteriorated microvascular flows irrespective of the presence or absence of the HO-1 induction. CONCLUSIONS: These results indicate that endogenous CO via HO-1 induction attenuates sevoflurane-induced microvascular endothelial interactions with leukocytes and platelets, although local nitric oxide levels appear to dominate microvascular flow in situ.

Endothelin B receptor-mediated protection against anoxia-reoxygenation injury in perfused rat liver: nitric oxide-dependent and -independent mechanisms.

This study aimed to investigate the roles of endothelin (ET) receptors in biliary dysfunction and cell injury in postischemic livers. Rat livers perfused with oxygenated Krebs-Henseleit solution were exposed to reoxygenation following 20-minute hypoxia. The anoxic perfusion decreased bile output and reduced cyclic guanosine monophosphate (cGMP) contents, an index of nitric oxide (NO) generation. Upon reoxygenation, the decreased bile was not fully recovered, and the resistance increased biphasically: an early transient spike accompanied by an elevated release of ET-1 and a rise accompanied by a cGMP elevation in the later period. The initial vasoconstriction appeared to be mediated by both ET(A) and ET(B) receptors, as judged by inhibitory effects of their antagonists, BQ-485 and BQ-788, respectively, while the late elevation of the resistance was not attenuated by these reagents, but rather enhanced by the ET(B) blockade. The BQ-788 treatment attenuated the reoxygenation-induced cGMP elevation and induced bile acid-dependent choleresis. However, such a change upon the ET(B) blockade coincided with dissociation of a recovery of phospholipids and aggravation of cell injury. The BQ-788-elicited deterioration of reoxygenation-elicited changes was attenuated by NO supplement with S-nitroso-N-acetyl penicillamine. N(omega)-Nitro-L-arginine methyl ester, an NO synthase inhibitor, mimicked biliary changes elicited by the ET(B) blockade but without causing notable cell injury. Under these circumstances, coadministration of clotrimazole, an inhibitor of cytochrome P450 mono-oxygenases, elicited the injury comparable with that observed under the ET(B) blockade. These results suggest that ET(B)-mediated signaling limits excessive bile acid excretion and plays a protective role against reoxygenation injury through mechanisms involving both NO-dependent and -independent processes.

Putidaredoxin-cytochrome P450cam interaction.

Cytochrome P450cam (P450cam) catalyzes the monooxygenation of D-camphor. During the enzymatic reaction, oxyferrous, D-camphor-bound P450cam forms a binary complex with reduced putidaredoxin as an obligatory reaction intermediate. We have found that reduced putidaredoxin undergoes EPR-detectable conformational changes upon formation of the intermediate complex and also upon formation of a binary complex with CO- or NO-ferrous, D-camphor-bound P450cam. The structural changes in putidaredoxin are almost identical irrespective of the ligand bound to P450cam, and distinct from and significantly larger than those induced by unliganded ferrous P450cam. The binary complex formation also induce conformational alterations in the CO- and NO-ferrous, D-camphor-bound P450cam, thereby evoking simultaneous changes in the structure of the two proteins. A molecular basis and roles of such structural changes in the D-camphor monooxygenation are discussed.

Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver.

BACKGROUND AND AIMS: Liver is a major organ for heme detoxification under disease conditions, but its self-protective mechanisms against the toxicity are unknown. This study aimed to examine roles of carbon monoxide (CO), the gaseous product of heme oxygenase (HO), in ameliorating hepatobiliary dysfunction during catabolism of heme molecules in endotoxemic livers. METHODS: Vascular resistance and biliary flux of bilirubin-IXalpha, an index of HO-derived CO generation, were monitored in perfused livers of endotoxemic rats. Livers were perfused with HbO(2), which captures nitric oxide (NO) and CO, or metHb, a reagent trapping NO but not CO. RESULTS: In endotoxin-pretreated livers where inducible NO synthase and HO-1 overproduced NO and CO, HbO(2) caused marked vasoconstriction and cholestasis. These changes were not reproduced by the NO synthase inhibitor aminoguanidine alone, but by coadministration of zinc protoporphyrin-IX, an HO inhibitor. CO supplementation attenuated the events caused by aminoguanidine plus zinc protoporphyrin-IX, suggesting that simultaneous elimination of these vasorelaxing gases accounts for a mechanism for HbO(2)-induced changes. This concept was supported by observation that metHb did not cause any cholestasis; the reagent captures NO but triggers CO overproduction through rapid degradation of the heme by HO-1. CONCLUSIONS: These results suggest protective roles of CO against hepatobiliary dysfunction caused by heme overloading under stress conditions.

Effects of a thiolate axial ligand on the pi-->pi* electronic states of oxoferryl porphyrins: a study of the optical and resonance Raman spectra of compounds I and II of chloroperoxidase.

Optical absorption and resonance Raman spectra have been investigated for enzymatic intermediates, compounds I and II, of chloroperoxidase (CPO) which contains a thiolate-ligated iron porphyrin. Compound I of CPO (CPO-I), an oxoferryl porphyrin pi cation radical, gave an apparently asymmetric single-peaked Soret band at 367 nm, for which band fitting analyses revealed the presence of two transition bands around 365 and 415 nm. Compound II of CPO (CPO-II), an oxoferryl neutral porphyrin, gave a split Soret spectrum with two bands (blue and red Soret bands) at 373 and 436 nm. Thus both CPO-I and CPO-II can be categorized as hyperporphyrins. The maximum extinction coefficients (epsilon(b) and epsilon(r)) and energies (Eb and Er) of the blue and red Soret bands of CPO-II were found to fall on an epsilon(b)/epsilon(r) versus Eb-Er correlation line derived from data reported for six-coordinate ferrous derivatives of cytochrome P450 and CPO. Corresponding data for CPO-I did not fall on the correlation line. Resonance enhancement of the FeIV=O stretching (vFeO) Raman band was found for CPO-I when Raman scattering was excited at wavelengths within both transition bands around 365 and 415 nm, while the vFeO Raman band was not identified for CPO-II at any of the excitation wavelengths examined here. These findings suggest that the thiolate axial ligand causes Soret band splitting of CPO-II through configuration interaction between the sulfur-->porphyrin e(g)* charge transfer and porphyrin a1u,a2u-->e(g)* transitions, while the FeO portion is important in determining the shape of the Soret band of CPO-I.

Altered expression of heme oxygenase-1 in the livers of patients with portal hypertensive diseases.

This study was designed to determine changes in expression of heme oxygenase (HO)-1, the stress-inducible and carbon monoxide-producing enzyme, in normotensive and portal hypertensive human livers. GTS-1, a monoclonal antibody against rat HO-1 cross-reacted with the human HO-1 and blocked its enzyme activity, allowing us to examine the activity and localization of HO-1. In controls, approximately 50% of the total HO activity was from HO-1 as judged by the sensitivity to GTS-1, while the rest of activity was from other isozymes such as HO-2. HO-1 was expressed mainly in a subpopulation of Kupffer cells, and the expression in hepatic stellate cells, sinusoidal endothelial cells, and hepatocytes was little, if any. The HO-1 expression exhibited quite different pictures in the livers of portal hypertensive diseases. In cirrhotic livers, which undergo portal hypertension through increases in intrasinusoidal resistance and regenerative changes in the parenchyma, HO-1 occurred in a majority of Kupffer cells and was also observed in hepatocytes. Consequently, the total HO-1 activities became significantly greater in these tissues than those from normal individuals. By contrast, livers of idiopathic portal hypertension that are characterized by an increase in presinusoidal resistance displayed a significant decrease in the HO-1 expression in Kupffer cells, and its hepatocellular expression was not detectable. Although factors involved in altered HO-1 expression in these cells remain unknown, the results suggest that Kupffer cells could alter their expression of HO-1 in response to local hemodynamic changes associated with chronic portal hypertension in humans.

Radioprotective effects of miso (fermented soy bean paste) against radiation in B6C3F1 mice: increased small intestinal crypt survival, crypt lengths and prolongation of average time to death.

The radioprotective effect of miso, a fermentation product from soy bean, was investigated with reference to the survival time, crypt survival and jejunum crypt length in male B6C3F1 mice. Miso at three different fermentation stages (early-, medium- and long-term fermented miso) was mixed in MF diet into biscuits at 10% and was administered from 1 week before irradiation. Animal survival in the long-term fermented miso group was significantly prolonged as compared with the short-term fermented miso and MF cases after 8 Gy of 60Co-gamma-ray irradiation at a dose rate of 2Gy min(-1). Delay in mortality was evident in all three miso groups, with significantly increased survival. At doses of 10 and 12 Gy X-irradiation at a dose rate of 4 Gy min(-1), the treatment with long-term fermented miso significantly increased crypt survival. Also the protective influence against irradiation in terms of crypt lengths in the long-term fermented miso group was significantly greater than in the short-term or medium-term fermented miso and MF diet groups. Thus, prolonged fermentation appears to be very important for protection against radiation effects.

X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center.

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.

Grafting of stomach tissue into the duodenum in F344 rats results in chimeric crypts and tumor development.

Gastric tissue was transplanted from the fundic and pyloric mucosa of 8-week old female F344 rats into the duodenum of males. Autopsy, 12 months after the operation, revealed grafts associated with persistent stones in the duodenum and/or calcification in the tissue. Pepsinogen positive chimeric glands with goblet cells also appeared in the grafts which gave rise to tumors in 18 out of 45 animals (40%). In conclusion, stomach grafts re-differentiate into intestine with goblet cells in the duodenum and this process predisposes to tumor development.

Formation of compound I in the reaction of native myoglobins with hydrogen peroxide.

Reaction of ferric native myoglobin (Mb) with hydrogen peroxide (H(2)O(2)) was studied by the aid of stopped-flow rapid-scan spectrophotometry. In contrast to the results in previous studies where compound I was reported to be undetectable, both sperm whale and horse heart metmyoglobins (metMbs) formed a significant quantity of compound I, an oxoferryl porphyrin pi-cation radical (Por(+)-Fe(IV)(O)), during their reactions with H(2)O(2). With both kinds of Mbs, formation of compound I was more clearly observed in D(2)O than in H(2)O. The compound thus formed was capable of performing monooxygenation of thioanisole to methyl phenyl sulfoxide and a 2-electron oxidation of H(2)O(2) giving O(2) and H(2)O as products. It was also converted into ferryl myoglobin (Por-Fe(IV)(O)-globin(+)) spontaneously. Rate constants for these reactions and that for a direct conversion of metMb to ferryl Mb through the homolysis of H(2)O(2) were determined. These results established unambiguously that native metMb can form both compound I and ferryl Mb upon reaction with H(2)O(2) and that these high valent iron compounds serve as essential intermediates in Mb-assisted peroxidative reactions. The observed deuterium effect on the apparent stability of compound I was attributable to that effect on the hydrogen abstraction step in the 2-electron oxidation of H(2)O(2) by compound I.

Synergistic induction of eotaxin expression in human keratocytes by TNF-alpha and IL-4 or IL-13.

PURPOSE: To investigate the effects of tumor necrosis factor (TNF)-alpha, interleukin (IL)4, and IL-13 on expression of the chemokine eotaxin by cultured human keratocytes. METHODS: Cultured human keratocytes were incubated with various combinations and concentrations of TNF-alpha, IL-4, and IL-13. The concentration of eotaxin in the culture supernatant was subsequently measured by enzyme-linked immunosorbent assay, and the amount of eotaxin mRNA in cell lysates was determined by reverse transcription-polymerase chain reaction analysis. RESULTS: Keratocytes incubated in the absence of cytokines did not release detectable amounts of eotaxin into the culture medium. Whereas incubation of keratocytes with TNF-alpha, IL-4, or IL-13 alone or with the combination of IL-4 and IL-13 had only a small effect on eotaxin release, exposure of the cells to TNF-alpha in combination with either IL-4 or IL-13 resulted in a marked increase in eotaxin production that was both time and dose dependent. The abundance of eotaxin mRNA in keratocytes was also increased in a synergistic manner by incubation of cells with TNF-alpha together with either IL-4 or IL-13. CONCLUSIONS: Stimulation of human keratocytes with the combination of TNF-alpha and either IL-4 or IL-13 resulted in synergistic increases in both the abundance of eotaxin mRNA and the release of eotaxin protein. This cytokine-induced increase in eotaxin production by keratocytes may contribute to eosinophil infiltration in inflammatory ocular diseases such as vernal keratoconjunctivitis.

Immunoneutralization of glycoprotein Ibalpha attenuates endotoxin-induced interactions of platelets and leukocytes with rat venular endothelium in vivo.

This study aimed to examine molecular mechanisms for endotoxin-induced adhesive changes in platelets in vivo. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester were visualized in rat mesenteric venules through intravital microscopy assisted by a high-speed fluorescence video imager at 1000 frames per second or by a normal-speed intensifier under monitoring of erythrocyte velocity. Leukocyte rolling was examined by normal-speed transmission video images. The velocity of platelets traveling along the centerline of venules followed that of erythrocytes, whereas that measured at the periendothelial space was significantly smaller than the erythrocyte velocity; a majority of these cells exhibited transient but notable rolling with endothelium. Administration of endotoxin increased the density of periendothelial platelets and reduced the rolling velocities of platelets and leukocytes in venules: All events were attenuated by anti-rat P-selectin monoclonal antibody s789G or by anti-human glycoprotein (GP) Ibalpha monoclonal antibody GUR83/35, which blocks ristocetin-induced aggregation of rat platelets. Isolated rat platelets injected into endotoxin-pretreated rats were able to roll on the venules. This event was attenuated by pretreatment of platelets in vitro with GUR83/35 but not with s789G, suggesting involvement of endothelial P-selectin and platelet GP Ibalpha in the endotoxin-induced responses. Furthermore, isolated human platelets showed similar rolling interactions with endotoxin-preexposed rat venules, and pretreatment of the platelets with GUR83/35, but not with s789G, significantly reduced such interactions. Our results provide the first evidence for involvement of GP Ibalpha in endotoxin-induced microvascular rolling of platelets and leukocytes, and this system serves as a potentially useful tool to examine GP Ibalpha-associated function of human platelets in vivo.

Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury.

We reported that carbon monoxide (CO) generated through heme oxygenase (HO) inhibits mitogen-induced proliferation of vascular smooth muscle cells (VSMCs). We report that balloon injury induces HO-1, the stress-inducible isozyme of HO, in VSMCs and inhibits neointimal formation through the action of endogenous CO. Northern blot analysis and immunohistochemistry revealed that HO-1 is markedly induced in the media as early as 1 day after injury, whereas only a little expression was detected in the intact carotid artery. The neointimal proliferative changes were augmented or inhibited by the HO inhibitors or inducer, respectively, and effects of these interventions were not altered by suppression of endogenous nitric oxide (NO), if any. To elucidate the mechanisms by which HO controls the proliferative changes, effects of alterations in the HO reaction were examined by determining angiotensin II-elicited VSMC proliferation in vitro: the HO inducer attenuated and its inhibitor restored the proliferative response to angiotensin II (1 nM and 100 nM). Hemoglobin, a reagent trapping both NO and CO, but not met-hemoglobin, which can capture NO but not CO, augmented the proliferative response. These data suggest that endogenous CO serves as a protective factor that limits the excessive VSMC proliferation associated with vascular diseases.

Stage-specific expression of mucosal addressin cell adhesion molecule-1 during embryogenesis in rats.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is essential for lymphocyte trafficking to gut-associated lymphoid tissues and is implicated in inflammatory disorders in the gut and pancreatic islets. In this study, we examined the functional role of MAdCAM-1 during rat ontogeny using newly generated specific mAb. As previously observed in mice and humans, MAdCAM-1 was preferentially expressed in high endothelial venules (HEV) in gut-associated lymphoid tissues and venules of lamina propria in adult rats. Lymphocyte rolling and adhesion on HEV in Peyer's patches (PP) were completely abrogated with neutralizing anti-MAdCAM-1 mAb, in agreement with the notion that MAdCAM-1 is the principal HEV ligand for lymphocyte rolling and adhesion in adult PP. In the developing gastrointestinal tract, MAdCAM-1 was widely expressed in the venules of the lamina propria of fetal rats. In addition, MAdCAM-1 was also expressed in follicular dendritic cells in the neonatal PP. Interestingly, MAdCAM-1 expression was found also in nonmucosal tissues during ontogeny. MAdCAM-1 was transiently expressed in blood vascular endothelial cells in the fetal skin and neonatal thymus. Notably, MAdCAM-1-positive blood vessels were localized mainly in the cortico-medullary junction in the neonatal thymus and about 10-20% of thymocytes, most of which were either CD4, CD8 double positive or single positive specifically reacted with soluble MAdCAM-1 via integrin alpha4beta7. After birth, MAdCAM-1 expression in thymus blood vessels disappeared and concomitantly, the soluble MAdCAM-1-reactive thymocytes were rapidly down-regulated. Our results suggest that MAdCAM-1 functions as a vascular addressin in not only mucosal, but also nonmucosal lymphoid tissues during ontogeny.

Daily regeneration of rat adrenocortical cells: circadian and zonal variations in cytogenesis.

Daily regeneration of rat adrenocortical cells were investigated in terms of circadian and zonal variations by following the cells at the DNA-synthesizing stage. An S-phase was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The BrdU-positive cells were observed throughout the day mainly in two regions of the adrenal cortex, i.e. the innermost portion of the zona glomerulosa and the outermost portion of the zona fasciculata. Cells only in a latter region showed a distinct circadian rhythm of cell proliferation with a peak at 3-4 a.m. A remarkable rise in the plasma adrenocorticotropin (ACTH) concentration preceded such an increase in the cell proliferation by about 4 hours. This phenomenon could be mimicked by raising the plasma ACTH concentration by the administration of Cortrosyn Z or metyrapone. Angiotensin II-stimuli induced by Na-deficiency increased the proliferation of zona glomerulosa cells in the former region at 6-7p.m without significant effects on that of the zona fasciculata cells in the latter region. Thus at least two sites, which respond differentially to the day/night cycle and circulating hormone levels, exist in rat adrenal cortex being responsible for the cytogenesis in this endocrine organ.

Studies on cytogenesis in adult rat adrenal cortex: circadian and zonal variations and their modulation by adrenocorticotropic hormone.

Circadian rhythms and zonal variations in the cell proliferation of adult rat adrenal cortex were studied by following the cells in the DNA-synthesizing stage (S-phase) as assessed by 5-bromo-2'-deoxyuridine incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The S-phase cells were observed throughout the day in two regions of the adrenal cortex: (i) a region from the inner half of the zona glomerulosa to near the outer margin of the zona fasciculata, and (ii) the outer one-fourth portion of the zona fasciculata. Very little change in number was observed in the former region between day and night, while a burst of cell proliferation occurred in early morning at 3-4 a.m. in the latter region. A prominent rise in the plasma adrenocorticotropic hormone (ACTH) concentration preceded the burst of cell proliferation by about 4 h. Upon raising the plasma ACTH concentration by administration of ACTH or metyrapone, prominent cell proliferation also occurred in the same portion of the zona fasciculata 4-6 h after the provoked ACTH surge. Thus at least two sites in rat adrenal cortex are responsible for cytogenesis in this endocrine organ, and respond differentially to day/night cycles and circulating ACTH levels.