Myristoylated alanine-rich C kinase substrate protein and mRNA in bovine corpus luteum during the estrous cycle

The bovine corpus luteum contains a myristoylated alanine-rich C kinase substrate (MARCKS) protein known to crosslink actin filaments in the cytoskeletal cortex associated with the plasma membrane. We conducted experiments to determine whether concentrations of MARCKS mRNA and protein in the bovine corpus luteum varied during theestrous cycle. Using Northern blots probed with a MARCKS cDNA, we found that luteal concentrations of MARCKS mRNA were greatest on d 4, 8, and 12 and markedly reduced on d 16 of the cycle (p<0.08). Similarly, Western blot analysis of luteal proteins revealed that concentrations of MARCKS protein were greatest on d 8 and least on d 16 of the cycle (p<0.01). Exposure of slices from a d 8 corpus luteum to prostaglandin F_2α (PGF_2α) during a 10-min incubation in the presence of [³²P]-orthophosphate resulted in enhanced phosphorylation of MARCKS in membrane and cytosolic fractions compared to that of controls. We therefore concluded that expression of the luteal MARCKS protein gene may be regulated and that PGF_2α-induced phosphorylation of this protein is attributable to activation of protein kinase C.     

Effect of losartan on angiotensin II-mediated endothelin and prostanoid excretion in humans

Angiotensin II (Ang II) stimulates renal prostanoid and vascular endothelin-1 (ET-1) release. Most known Ang II effects are mediated by AT₁ receptors. Our aim was to determine whether AT₁ receptor activation mediates Ang II-evoked renal prostanoid and ET-1 release. Eleven healthy men were randomized in a crossover, double-blind fashion to receive 100 mg/day of losartan or matching placebo, for 8 days. Blood and urine were sampled before and after a 2-h infusion of Ang II at a rate previously determined to increase mean arterial pressure (MAP) by 25 to 30 mm Hg in each subject. After a 14-day washout, subjects received the alternate treatment. Pretreatment with losartan had little effect on baseline MAP, but increased plasma renin activity, and virtually eliminated the pressor response to Ang II infusion. Angiotensin II significantly increased prostanoid excretion after placebo; the prostanoid response to Ang II was even greater after losartan. Plasma ET-1 was not altered by Ang II infusion, with or without losartan. In contrast, urine ET-1 excretion rate decreased to 40% of baseline after Ang II but not after losartan pretreatment; losartan alone had no effect. We conclude that Ang II decreases renal ET-1 synthesis and release through the AT₁ receptor. In contrast, Angiotensin II-mediated renal prostanoid synthesis does not require activation of AT₁ receptors. These findings indicate that AT₁ receptor antagonists could provide renal protection through indirect mechanisms.     

Mechanism of nucleotide inhibition of gonadotropin binding to cell membranes of bovine corpus luteum.

ATP, CTP, ADP, AMP, cyclic 3',5'-AMP (cAMP) and cyclic 3',5'-CMP (cCMP) effectively inhibited the specific binding of 125I-labelled human chorionic gonadotropin ([125I]HCG) to bovine corpus luteum cell membranes. This inhibition was observed with 2.5 X 10(-4) M to 1.0 X 10(-3) M nucleotide concentrations, regardless of the presence of a nucleotide regenerating system. Submaximal concentrations of combinations of the nucleotides were additive in inhibiting binding. The inhibition of [125I]HCG binding was observed when the nucleotides were added at the beginning of or during incubation or preincubation of the membranes with nucleotides. Preincubation of membranes with CTP and cAMP, subsequent washing and reincubation with hormone, showed time-dependent inhibition of [125I]HCG binding when the preincubation temperature was 38 degrees C but not at 4 degrees C. The concentrated supernates from nucleotides preincubated with membranes had no inhibitory effect on [125I]HCG binding to fresh membranes. In the absence of added nucleotides, [125I]HCG-membrane interaction had the following apparent binding constants: a Kd of 1.5 X 10(-10) M, 46.3 fmoles of binding sites per mg membrane protein, and rate constants for association and dissociation 4.0 X 10(6) M-1 sec-1 and 1.0 X 10(-3) sec-1, respectively. At steady state conditions of [125I]HCG binding, CTP inhibited [125I]HCG at lower concentrations of added hormone (less than 3 X 10(-9) M) whereas at higher concentrations, this nucleotide enhanced [125I]HCG binding. Scatchard analysis of the data revealed that inhibition and enhancement of [125I]HCG binding in the presence of CTP were due to lowered affinity of gonadotropin receptors (32-37) fold) and to exposure of new low-affinity binding sites for [125I]HCG, respectively. At non-steady-state conditions, nucleotides increased dissociation rates (80 to 100%) and decreased association rates (30 to 38%). The data appear to be compatible with the suggestion that the nucleotides may bind to sites in the membranes and subsequently induce conformational changes in membrane components, resulting in a decreased affinity of gonadotropin receptors. The physiological significance of these findings needs to be determined.     

Identification and quantitative determination of steroids in bovine corpus luteum during oestrous cycle and pregnancy.

Neutral steroids in bovine corpus luteum were isolated by liquid-gel chromatography on hydrophobic Sephadex, and were analyzed by computerized gas chromatography-mass spectrometry. The presence of progesterone and 20beta-hydroxy-4-pregnen-3-one was confirmed. In addition, 3beta-hydroxy-5-pregnen-20-one, 5-pregnene-3beta,20beta-diol, 3beta-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnane-3beta,20beta-diol were fully identified, and 3-hydroxy-4-pregnen-20-one, 4 pregnene-3,20-diol, 22-hydroxycholesterol and 20,22-dihydroxycholesterol were partially characterized. Steroid sulphates were not detected. Quantification of the six fully identified steroids was based on peak areas in specific fragment ion current chromatograms constructed by the computer. During the 4th-19th day of the oestrous cycle the steroid concentrations varied as follows: progesterone 6.0-36.7 mug/g wet luteal weight, 20beta-hydroxy-4-pregnen-3-one 0.8-5.5 mug/g, 3beta-hydroxy-5-pregnen-20-one 1.0-7.1mug/g, 5-pregnene-3beta,20beta-diol smaller than 0.2-0.9 mug/g, 3beta-hydroxy-5alpha-pregnan-20-one 1.7-8.6 mug/g, and 5alpha-pregnane-3beta,20beta-diol smaller than 0.2-1.2 mug/g. The concentrations of progesterone and 3beta-hydroxy-5-pregnen-20-one seemed to vary in parallel and were low during days 11-17. During this period the concentrations of 5-pregnene-3beta,20beta-diol and 5alpha-pregnane-3beta,20beta-diol were highest as was the relative contribution of all three 20beta-hydroxysteroids to the total amount of steroids. The relative amount of 3beta-hydroxy-5alpha-pregnan-20-one seemed to be highest during days 4-6. The total steroid concentration in corpora lutea taken in early pregnancy (75-105 days) was 18-47 mug/g. In the period 75-90 days, progesterone constituted only 35-42% of the total steroids, 3beta-hydroxy-5alpha-pregnan-20-one as much as 23-40% and the total 20beta-hydroxysteroids 18-30%. The total steroid concentration in corpora lutea taken in midterm and late pregnancy was 21-77 mug/g. In this period progesterone was by far the predominant steroid and constituted about 80-90% of the total steroids in corpora lutea taken between days 150 and 240. Possible correlations between luteal growth, steroid oxidoreductases and steroid concentrations are discussed.     

Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy

Administration of RU486 to late pregnant rats results in preterm delivery 24 h after treatment and the induction of a luteolytic process after labor. We investigated whether functional changes occurring within the corpora lutea after RU486 treatment were associated with morphologic features of apoptotic cell death. Rats on d 18 of pregnancy were treated with RU486 (5 mg/kg) at 10:00 am and killed 72 h after. We studied the number of apoptotic cells in paraffin sections of the corpora lutea by routine hematoxylin and eosin (H&E) staining, and by in situ 3′ end labeling (TdT-mediated dUTP nick-end labeling [TUNEL]). The corpora lutea were also processed for electron microscopy to study ultrastructural changes after RU486 treatment. The number of cells showing apoptotic nuclei in H&E-stained sections was higher in RU486-treated animals than in controls (vehicle-treated rats). The quantification of the number of apoptotic nuclei within the corpora lutea performed by TUNEL confirmed the higher number of apoptotic nuclei in animals receiving the antigestagen compared with controls. Ultrastructurally, the luteal cells undergoing a poptosis presented a highly deteriorated cytoplasmic organization The nuclei, in an initial step of regression, displayed condensation of the chromatin, a prominent nucleolus, and a perinuclear space. In an advanced step of degeneration, the nuclei showed evidence of large irregular aggregates of condensed chromatin. Prostaglandin F_2α(PGF_2α), which mediates the luteolytic action of RU486, mimicked the effect of the antigestagen on the induction of apoptosis when administered to rats on d 18 of pregnancy (100 μg at 9:00 am and 1:00 pm), which were killed 72 after the last injection. In conclusion, the present results indicate that functional luteolysis in rats is associated with structural luteal regression with the morphologic features of apoptotic cell death, as demonstrated by studying the luteolytic process induced by the administration of the antigestagen RU486.     

Opposite effects of ethanol on the activation of adenylyl cyclase in human corpus luteum membranes

The influence of an acute exposure to ethanol on adenylyl cyclase activity in membrane fractions prepared from human corpus luteum was investigated. Ethanol up to a concentration of 5% (v/v) was without effect on basal luteal adenylyl cyclase activity, but markedly potentiated stimulation of NaF and hCG in a dose-dependent manner. In contrast, ethanol progressively inhibited forskolin stimulation at the same range of ethanol concentrations. Maximal NaF and hCG responsiveness of adenylyl cyclase activity was observed at 5% ethanol and reached values 80% and 100% higher than controls without ethanol, respectively. However, at the same ethanol concentration, forskolin-stimulated enzymatic activity was reduced by 40% relative to controls. Equilibrium binding studies involving [¹²⁵I]hCG interaction with luteal membranes in the presence of the concentration of ethanol showing maximal hCG responsiveness indicated that ethanol slightly affected (15% increase) the hCG binding compared to controls, without any appreciable change on the K_d for the hormone. This minor effect of ethanol on gonadotropin binding sites contrasted greatly with the extent at which ethanol maximally potentiated the gonadotropin-stimulated adenylyl cyclase. GTP was found to be less effective than GMP-P(NH)P in sustaining ethanol potentiation, suggesting that ethanol is unlikely to act by inhibiting GTPase activity. These data indicate that the acute effects of ethanol inhibit forskolin-stimulated adenylyl cyclase at concentrations potentiating stimulatory effects of NaF and of hCG, and that the synergistic interaction of ethanol and gonadotropin stimulation of adenylyl cyclase is, at least in part, due to an increase in the functional coupling of the occupied hCG-receptor complex with the components of the enzyme system.     

Abundant expression of sodium-potassium-activated adenosinetriphosphatase alpha 1 subunit in corpus luteum of porcine ovary

Follicular development is accompanied by the accumulation of follicular fluid. During corpus luteum formation, follicular fluid is diminished and antrum is replaced by lutein cells. These dynamic changes in fluid distribution suggest the existence of control mechanism of fluid transport and membrane permeability. One of the major factors regulating membrane permeability is the sodium-potassium-activated adenosinetriphosphatase (Na⁺-K⁺-ATPase). To elucidate the possible involvement of Na⁺-K⁺-ATPase in follicular growth and luteinization, immunohistochemical localization of Na⁺-K⁺-ATPase α1 subunit and enzyme activity in porcine ovary were investigated. In primordial follicles, Na⁺-K⁺-ATPase α1 subunit immunostaining was localized only in the oocyte and the surrounding stromal cells. In preantral follicles, immunostaining for Na⁺-K⁺-ATPase α1 subunit became apparent in granulosa and theca cells. As the follicle matured, the staining intensity in the oocyte, theca, and granulosa cells increased, which corresponded with the enzyme activity. Na⁺-K⁺-ATPase α1 subunit immunostaining became most abundant in granulosa and theca lutein cells in corpus luteum, and decreased in the regressing corpus luteum. Enzyme activity in corpus luteum was significantly higher than that in the follicles. This is the first study indicating that Na⁺-K⁺-ATPase α1 subunit expression is augmented in granulosa cells by follicular growth and most abundant in lutein cells in the corpus luteum, suggesting its possible involvement in corpus luteum formation.     

The effect of corpus luteum on hormonal composition of follicular fluid from different sized follicles and their relationship to serum concentrations in dairy cows

ObjectiveTo investigate the effect of the presence or absence of corpus luteum on hormonal composition of follicular fluid (FF) from different sized follicles and their relationship to serum concentrations in dairy cows.MethodsOvaries were collected from 30 clinically healthy adult female cows (Holstein Friesian) 4–7 years of age with clinically normal reproductive tracts after slaughtering. Blood samples were collected from the jugular vein before slaughter from each cow. The stage of the cycle in the cows was determined postmortem. The ovaries collected from per cow were classified with corpus luteum (CL⁺) and without corpus luteum (CL⁻). FF was aspirated from small (3-5 mm), medium (6-9 mm), and large (10-20 mm) follicles in CL⁺ and CL⁻ ovaries. Serum and FF samples were analyzed for estradiol-17β, progesterone, testosterone, T₃ and T₄ concentrations.ResultsResults demonstrated that the FF concentrations of estradiol-17β, progesterone and testosterone in different sized follicles categories (small, medium and large follicles in CL⁺ and CL⁻ ovaries) were significantly higher (P≤0.05) when compared with the serum. The FF concentrations of estradiol-17β and testosterone in same follicle size categories in CL⁺ and CL⁻ ovaries were also significant (P<0.05). Indeed, concentrations of these hormones in the CL⁻ ovaries were higher than those of the CL⁺ ovaries. However, there was a statistically significant difference between medium and large follicles for progesterone concentration in CL⁺ and CL⁻ ovaries (P<0.05). There was a significant correlation between concentration of hormones in serum and FF with increased follicular diameter.ConclusionsThese results indicated that the levels of hormonal composition in the FF were related to follicular size and interestingly to the presence or absence of a corpus luteum. Indeed, the corpus luteum locally affects neighboring follicular compositions during the luteal phase of the estrous cycle in dairy cows.     

Angiogenesis in the human corpus luteum: changes in expression of angiopoietins in the corpus luteum throughout the menstrual cycle and in early pregnancy

CONTEXT: Blood vessel stabilization is regulated by angiopoietins and important for angiogenesis in the corpus luteum. OBJECTIVE: To study angiogenesis and blood vessel stabilization in the human corpus luteum, changes in expression of angiopoietin (Ang)-1, Ang-2, and their specific receptor, Tie-2, together with the number of blood vessels and pericytes were examined in the corpus luteum throughout the menstrual cycle and in early pregnancy. DESIGN: The number of blood vessels and pericytes was determined by immunohistochemistry for CD34 and alpha-smooth muscle actin, respectively. Ang and Tie-2 expression were examined by immunohistochemistry or RT-PCR. RESULTS: The number of blood vessels increased during the early luteal phase, whereas the number of pericytes was small in the early luteal phase and increased in the midluteal phase, suggesting that angiogenesis is undergoing during the early luteal phase and blood vessels are stabilized in the midluteal phase. Blood vessels and pericytes decreased in number during the late luteal phase. The increased number of both blood vessels and pericytes seen in the corpus luteum of early pregnancy suggests that angiogenesis is undergoing accompanied by blood vessel stabilization. Ang-2 expression with low Ang-1 expression was found during the early luteal phase. Thereafter, increasing Ang-1 expression during the midluteal phase, declining Ang-1 expression with continued Ang-2 expression during the late luteal phase, and relatively high Ang-1 expression in early pregnancy were observed. CONCLUSIONS: The change in Ang expression is closely associated with angiogenesis, blood vessel stabilization, and blood vessel regression during the divergent phases of luteal formation, luteal regression, and luteal rescue by pregnancy.     

原发性高血压患者血管紧张素转化酶和血管紧张素受体基因多态性的研究

目的　探讨血管紧张素转化酶 ( ACE)及血管紧张素 - 1型受体 ( AT1 R)基因多态性与原发性高血压 ( EHT)的关系。方法　应用聚合酶链反应及 PCR加酶解方法检测 1 50例健康人 ( NT)及 1 52例 EHT患者 ACE I/ D基因多态性的 ACE及 AT1 R A1 1 6 6 C突变。结果　 EHT组ACE I/ D基因多态性等位基因频率 I为 0 .50 ,D为 0 .50 ,D等位基因频率及基因型频率显著高于 NT组 ( P<0 .0 5) ;而两者之间的 AT1 R A1 1 6 6 C的C等位基因频率差异无显著性 ( P>0 .0 5)。结论　 ACE基因可能是 EHT的重要遗传因素 ,AT1 R基因 A1 1 6 6 C多态性与 EHT无关     

Effects of angiotensin II and angiotensin II type 1 receptor blockade on neointimal formation after stent implantation

The prevalence of chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF) increases substantially with age. The coexistence of COPD and CHF is common but often unrecognized in elderly patients. To avoid overlooking COPD in elderly patients with known CHF pulmonary function tests should be routinely obtained. Likewise, to avoid overlooking CHF in elderly patients with known COPD left ventricular (LV) function should be routinely assessed. Plasma brain natriuretic peptide levels are useful to differentiate COPD exacerbation from CHF decompensation in patients presenting with acute dyspnea. Aging exacerbates skeletal muscle alterations that occur in patients with CHF and COPD. Skeletal muscle metabolic alterations and atrophy and the resulting deterioration of functional capacity progress rapidly in elderly patients with COPD and CHF. Physical conditioning reverses rapidly progressing skeletal muscle metabolic alterations and atrophy and promotes independence and life quality in the elderly. Physical conditioning is clearly an essential component of the management of elderly patients with COPD and CHF. The pharmacological management of patients with coexistent COPD and CHF should focus on not depriving these patients from long-term beta adrenergic blockade. Long-term beta adrenergic blockade has been repeatedly shown to improve survival in elderly patients with CHF due to LV systolic dysfunction and, contrary to conventional belief, is well tolerated by patients with COPD.     

Down-regulation of cardiac apelin system in hypertrophied and failing hearts: Possible role of angiotensin II-angiotensin type 1 receptor system

Cardiac apelin has recently been suggested to contribute to the pathophysiology of heart failure (HF) in humans. In animal experiments, its infusion acutely improved systolic as well as diastolic LV function. Although its deficit could critically determine the cardiac dysfunction, its regulatory mechanism is unknown. Accordingly, we investigated the role and regulation of the cardiac apelin system in the diseased heart using Dahl salt-sensitive rats, which show a distinctive transition from compensatory LV hypertrophy (LVH) to HF. In the compensatory LVH stage, apelin and its receptor APJ mRNA showed no change compared with control animals, while these were markedly down-regulated in the HF stage (72% and 57% decrease, respectively). The rats were chronically treated with telmisartan (angiotensin type 1 receptor blocker [ARB], 5 mg/kg/day, n=9), ONO-4817 (matrix metalloproteinase [MMP] inhibitor, 200 mg/kg/day, n=9), bisoprolol (beta blocker, 3 mg/kg/day, n=6) or vehicle (0.5%CMC, n=9) from the LVH stage. Although the functional improvements were similar among the three treated groups 6 weeks after treatment, restoration of cardiac apelin and APJ expression was observed only in the ARB group. Furthermore, in angiotensin II-infused rats, cardiac apelin mRNA was decreased after 24 h of treatment and its restoration was achieved by treatment with ARB. These results indicate that the cardiac apelin system is markedly down-regulated in experimental HF and may be regulated by the angiotensin II-angiotensin type 1 receptor system directly. Inhibition of the renin-angiotensin system may have beneficial effects, at least in part, through restoration of the cardiac apelin system in the treatment of HF.     

Inhibition of angiotensin Ⅱ and blockade of endothelin receptors reduce arterial calcification in rats

Objective To examine whether the two vascular paracrine/autocrine factors, angiotensin Ⅱ (Ang Ⅱ) and endothelin, participate in the pathogenesis of arterial calcification. Methods Nicotine and vitamin D3 treated rats were studied. Vascular calcification was confirmed by using Von Kossa staining, measurement of calcium content,45Ca2+ uptake assay and alkaline phosphatase (ALP) activity. The plasma and vascular Ang Ⅱ and endothelin levels were measured by using radioimmunoassay. Angiotensinogen and endothelin mRNA levels were determined by RTPCR. Results The arterial calcium content, 45Ca2+ uptake and ALP activity were increased in calcification groups compared with control ( P ＜ 0.01 ). Administration of the angiotensin receptor antagonist losartan, the endothelin receptor antagonist bosentan, and the angiotensin-converting enzyme inhibitor captopril reduced significantly the arterial calcium content, 45Ca2+ uptake and ALP activity. In addition, the plasma and aortic Ang Ⅱ and endothelin contents, and vascular angiotensinogen and endothelin mRNA expression were significantly up-regulated ( P ＜0.05).Conclusions These findings suggest that functional renin-angiotensin system and endothelin pathway are involved in vascular calcification, and that activation of these systems could potentiate pathogenesis of arterial calcification. ( J Geriatr Cardiol 2004;1(2) :108-113. )     

胰腺癌细胞系血管紧张素Ⅱ1型受体蛋白表达的研究

目的：探讨血管紧张素Ⅱ1型受体（angiotensin Ⅱ type 1 receptor,AT1）在胰腺癌细胞中的表达。方法：用免疫细胞化学染色检测胰腺癌细胞系SW1990、PaTu8988s和PANC－1中AT1蛋白的表达。结果：胰腺癌细胞系SW1990、PaTu8988s和PANC－1中均有AT1蛋白的表达，结论：AT1在胰腺癌细胞生长中可能发挥重要作用，应用AT1拮抗剂对防治胰腺癌似乎有一定意义。