Immunocytochemical localization of pro gamma MSH, gamma MSH, ACTH and beta endorphin/beta lipotrophin in the fetal sheep pituitary: an ontogenetic study

An immunocytochemical staining technique was used to localize four fragments [pro gamma MSH, gamma MSH, ACTH and beta endorphin/beta lipotrophin (beta endorphin/beta LPH)] of the proopiomelanocortin molecule in both the adult and fetal sheep pituitary. In the adult sheep anterior pituitary each fragment was localized in cells that were darkly stained, stellate and widely distributed throughout the gland. The same cells, identified in three serial sections, stained with anti-pro gamma MSH, anti-ACTH and anti-beta endorphin/beta LPH. In the fetal sheep anterior pituitary all the proopiomelanocortin derived fragments were present at 38 days gestation. Between about 90 and 130 days of gestation both adult type proopiomelanocortin cells (small, stellate) and uniquely fetal cells (large, columnar) were present. Both adult-type and fetal proopiomelanocortin cells were identified in serial sections of the fetal anterior pituitary, stained with anti-pro gamma MSH, anti-ACTH and anti-beta endorphin/beta LPH. The adult intermediate lobe was immunoreactive with anti-pro gamma MSH and anti-beta endorphin/beta LPH but not with anti-gamma MSH or anti-ACTH. The fetal intermediate lobe was immunoreactive with all four antisera from 60 days gestation.     

Angiotensin II mediated acth release in rat pituitary cell culture

Anterior pituitaries from normal rats were enzymatically dispersed and placed into monolayer cell culture in order to determine if and how angiotensin II (Ang II) mediates the $\text{in vitro}$ release of ACTH and other pituitary hormones. Ang II stimulated ACTH secretion in a time dependent fashion. This release occurred at physiologic concentrations of Ang II and was linearly correlated with the log dose of Ang II. One hour pretreatment of the cells with cycloheximide, a inhibitor of protein synthesis, significantly decreased the cellular ACTH secretory response to Ang II. Ang 11 did not mediate the release of LH nor of ADH, a proposed stimulator of ACTH secretion.     

Effect of sodium valproate on the secretion of proopiomelanocortin derived peptides from cultured rat anterior pituitary cells

We examined effects of sodium valproate, a gamma amino butyric acid (GABA)-transaminase inhibitor, on the secretion of immunoreactive (IR)-ACTH and IR-beta-endorphin/LPH from cultured rat anterior pituitary cells to determine whether sodium valproate has a direct action on the secretion of ACTH and its related peptides from the cultured rat anterior pituitary gland. During the 3 h incubation, the basal secretion of IR-ACTH and IR-beta-endorphin/LPH decreased to 50.8% and 58.3%, respectively, of the control concentration after adding 10(-7) M sodium valproate into the incubation media and to 67.7% and 69.3%, respectively, of the control levels with 10(-8) M sodium valproate. However, sodium valproate at a concentration of 10(-6) M or 10(-9) M did not affect the basal concentration of IR-ACTH and IR-beta-endorphin/LPH. Sodium valproate at a concentration of 10(-7) M significantly attenuated the stimulated release of IR-ACTH and IR-beta-endorphin/LPH by 10(-9) or 10(-10) M of ovine corticotrophin releasing factor. These results indicate that sodium valproate could directly effect rat anterior pituitary cells to suppress both basal and stimulated release of proopiomelanocortin derived peptides and this supports the hypothesis that sodium valproate has a direct effect at the pituitary corticotroph in reducing plasma ACTH.     

The Ang II-induced growth of vascular smooth muscle cells involves a phospholipase D-mediated signaling mechanism

Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.     

Angiotensin II Promotes Leptin Production in Cultured Human Fat Cells by an ERK1/2‐dependent Pathway

Objective: The fat cell hormone leptin is known to be implicated in the pathogenesis of hypertension and cardiovascular disease. Here we tested whether angiotensin (Ang) II is involved in the control of leptin release from human adipocytes. Research Methods and Procedures: Leptin secretion was assessed from in vitro differentiated human adipocytes by radioimmunoassay. Western blot experiments were used to test for the signaling pathway activated by Ang II. Results: Ang II increased leptin secretion into the culture medium in a dose‐ and time‐dependent fashion. At 10^?5 M Ang II, the leptin concentration in the medium was increased at 24 hours by 500 ± 222% compared with control cultures (p < 0.05). This effect was also seen at the mRNA level. Similar effects were seen after exposure of fat cells to Ang III and Ang IV. Preincubation of fat cells with candesartan, an angiotensin II type 1 receptor antagonist, or the extracellular‐signal‐regulated kinases 1 and 2 inhibitor UO126 completely abolished the effect of Ang II on leptin production. The peroxisome proliferator‐activated receptor‐gamma agonist troglitazone modestly attenuated leptin release. Discussion: In conclusion, Ang II and its metabolites stimulated leptin production in human adipocytes. This effect is mediated through an extracellular‐signal‐regulated kinases 1 and 2‐dependent pathway and includes the angiotensin II type 1 receptor subtype.     

Partial characterization of a glycoprotein comprising the NH2-terminal region of mouse tumor cell pro-adrenocorticotropic hormone/endorphin.

The glycoprotein accounting for most of the nonadrenocorticotropic hormone (ACTH), non-beta-lipotropin (beta LPH) region of mouse tumor cell pro-ACTH/endorphin was purified from tumor cell culture medium and shown to contain 1/2 cystine residues. Preparations of the 16,000-dalton fragment-related material (referred to as 16K fragment) were heterogeneous with respect to size and charge. Despite this heterogeneity, a partial amino acid sequence for the NH2-terminal region of the molecule was determined by automated Edman degradationof the 16K fragment labeled by reduction and alkylation with [3H]iodoacetic acid or labeled biosynthetically with [3H]tryptophan. The sequence of 1/2 cystine and tryptophan residues in the mouse tumor 16K fragment can be aligned with one region of the amino acid sequence predicted from the cDNA for a bovine precursor to ACTH/beta LPH (Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y., Cohen, S.N., and Numa, S. (1979) Nature 278, 423--427).     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的心脏成纤维细胞增殖中的作用

本研究观察了钙调神经磷酸酶（ＣａＮ）在血管坚张素Ⅱ（ＡｎｇⅡ）刺激的大鼠心脏成纤维细胞增殖中的作用。在培养的大鼠心脏成纤维细胞上,应用双波长荧光 计检测Ｆｕｒａ－２标记的细胞游离Ｃａ＾２＋浓度;应用对硝基苯磷酸（ＰＮＰＰ）作底物测定钙调神经磷酸酶（ＣａＮ）活性;根据＾３Ｈ－胸腺嘧啶掺入法评估ＣａＮ特异性抑制剂环胞素Ａ（ＣｓＡ）对ＡｎｇⅡ刺激的心脏成纤维细胞ＤＮＡ合成的影响。结果表明,ＡｎｇⅡ（１０     

Steroidogenic activity of high molecular weight forms of corticotropin.

The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules     

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.     

Oxidant injury increases cell surface receptor binding of angiotensin II to pulmonary artery endothelial cells

Nitrogen dioxide (NO2), an environmental oxidant, is known to activate phospholipase A1 and modulate the plasma membrane structure of porcine pulmonary artery endothelial cells. We evaluated the effects of exposure to NO2, purified phospholipase B (which acts as phospholipase A1 and A2), or phospholipase A2 on 125I-angiotensin II (Ang II) receptor binding, internalization, or both in pulmonary endothelial cells. Exposure to 5 ppm NO2 for 48 hr at 37 degrees C or 0.075 U each of phospholipase B or A2 in phosphate-buffered saline (PBS) for 30 min at 24 degrees C resulted in an increase in total Ang II binding (i.e., cell surface bound and internalized) by 45% (p less than 0.05), 50% (p less than 0.05), and 85% (p less than 0.001), respectively, compared to controls. An Ang II receptor antagonist, [Sar1 Ile8] Ang II, competitively displaced Ang II binding to control, NO2-, phospholipase B-, and phospholipase A2-exposed cells. Dissociation of bound Ang II in the presence of PBS was less than 1% of total bound Ang II in control, NO2-, and phospholipase B-exposed cells and was 50% of total bound Ang II in phospholipase A2-exposed cells. In the presence of isotonic acetic acid/NaCl, in excess of 90% of cell surface-bound Ang II was dissociated from control, NO2-, and phospholipase B-exposed cells, and there was less than 2% of Ang II detectable when acid-treated cells were subjected to NaOH solubilization. In cells exposed to phospholipase A2, acetic acid treatment did not release cell-bound Ang II, and the remaining Ang II was recovered in the NaOH solubilized fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different patterns of central and peripheral beta EP, beta LPH and ACTH throughout life

The aim of the present study was to evaluate the relationship between central and peripheral concentrations of proopiocortin-related peptides in different periods of life. One hundred and eighty-nine plasma samples from normal subjects (18-87 years) obtained in basal conditions, and 20 cerebrospinal fluid (CSF) samples obtained by lumbar puncture from healthy volunteers (18-75 years) were studied. beta Lipotropin (beta LPH), beta endorphin (beta EP) and ACTH were measured by specific RIA after silicic acid plasma extraction and gel chromatography (beta LPH and beta EP). No sex differences were found in the patterns of the three peptides either in the plasma or in CSF. In the plasma samples, both beta LPH and beta EP concentrations showed a pattern throughout life which was expressed by a paraboloid function with the lowest values found in young and old subjects and with peaks at 51.3 and 48.2 years, respectively. On the contrary, ACTH values failed to be represented by a significant linear or curvilinear regression and presented only a slight decrease in subjects over 75 years of age. CSF levels of beta LPH were significantly lower in 45-76 year old subjects (18.8 +/- 12.6 fmol/ml, M +/- SD) than in 18-44 year old subjects (34.5 +/- 15.8; p less than 0.05), as were those of beta EP (elderly: 41.2 +/- 19.7; young: 94.2 +/- 36.7; p less than 0.05), which showed a significantly linear inverse correlation with age (r = 0.6062, p less than 0.01). These CSF samples did not show any ACTH variations connected with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potassium Depletion and Regulation of Angiotensin II Receptors in Vascular Smooth Muscle Cells

Abstract

Mesenteric artery smooth muscle cells were grown in culture media containing high, normal, or low concentrations of potassium to study the effects on angiotensin II (Ang II) receptor regulation. Cell growth was similar among cells grown in the different culture media. Cells grown in high potassium media (K=5.8 mEq/L) had an equilibrium dissociation constant, K_d, of 1.59 ± 0.2 nM, whereas those grown in normal potassium media (K=4.1 mEq/L) had a K_d of 1.79 ± 0.2 nM and those grown in a low potassium media (K=2.9 mEq/L) had a K_d of 1.19 ± 0.12 nM (not significantly different, NS). Binding capacity of smooth muscle cells grown in high potassium media was 81 ± 16.7 fmol/mg prot, 95.1 ± 12.4 fmol/mg prot in those grown in normal potassium media and those grown in low potassium media 86.4 ± 24.1 fmol/mg prot (NS). Binding of radiolabelled Ang II was reduced by approximately 70% in cells exposed to unlabelled Ang II for 30 or 60 minutes. However, this effect of exposure to Ang II to reduce subsequent binding of Ang II was identical in cells grown in high and low potassium medium. Therefore, we were unable to identify a direct effect of low potassium to induce changes in Ang II receptor binding affinity or binding capacity. Previously observed changes in these Ang II binding parameters in potassium-depleted rats was probably a consequence of other factors which were simultaneously altered by potassium deficiency.     

Lack of alpha-1-adrenergic receptor-mediated downregulation of angiotensin II receptors in neuronal cultures from spontaneously hypertensive rat brain

Neuronal cells from Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rat brains were established in culture to compare the expression of angiotensin II (Ang II) specific receptors and their regulation by norepinephrine (NE). Neurons from SH rat brains possess twice more Ang II specific receptors and expressed a proportional increase in Ang II stimulated [³H]-NE uptake compared with WKY neurons. NE caused a dose-dependent decrease in¹²⁵I-Ang II binding in WKY neurons, an effect not observed when neurons from SH rat brains were incubated with NE. These observations suggest that the lack of NE-induced downregulation of Ang II receptors in neuronal cultures is genetically regulated.     

Angiotensin II interacts with prostaglandin F2alpha and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro

Recent findings suggest that the ovarian renin-angiotensin system may regulate ovarian function through the paracrine/autocrine actions of angiotensin II (Ang II). In this study, we have examined and characterized the local effects of Ang II as a luteolytic factor and its interaction with prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1) in the bovine corpus luteum (CL) of the mid-luteal phase, by using an in vitro microdialysis system (MDS). Ang II was detected in the MDS perfusate (4 pg/ml), and infusion of PGF2alpha (10(-6) M) for 2 h increased the Ang II release by 50-100% during the following experimental period, in addition to its stimulation of ET-1 release. Two 2-h infusions of Ang II (10(-7)-10(-5) M) separated by a 2-h interval induced a dose- and time-dependent decrease of progesterone (P4) release by 41-66%. When the luteal explants were pre-perfused with PGF2alpha (10(-6) M) for 2 h, two consecutive perfusions of Ang II (10(-6) M) at a 2-h interval rapidly reduced the P4 release (by 50%). This reduction occurred 6 h earlier than those of infusions of PGF2alpha or Ang II alone. The simultaneous infusion of either 1) Ang II (10(-6) M) with PGF2alpha (10(-6) M), 2) ET-1 (10(-7) M) with PGF2alpha, or 3) Ang II + ET-1 with PGF2alpha (10(-6) M) for 2 h also induced a rapid and pronounced (60%) decrease in P4 release. Perfusion with the Ang II antagonist blocked the P4-suppressing activity of Ang II alone or PGF2alpha + Ang II infusion. Ang II stimulated the release of ET-1 and oxytocin during infusion but inhibited them after infusion. These results show that Ang II is released in the bovine midcycle CL in vitro, and this peptide, either alone or together with PGF2alpha, can suppress the release of P4. As PGF2alpha directly stimulated Ang II release, Ang II may influence the critical period for starting the cascade of functional luteolysis in vivo and might lead to structural luteolysis with ET-1 as a major vasoconstrictor. The overall results suggest that Ang II may have an important role at luteolysis in the bovine CL.     

pp60c-src在血管平滑肌细胞内丝裂原活化蛋白激酶激活中的作用

观察ｐｐ６０ｃ－ｓｒｃ在血管紧张素Ⅱ（ＡｎｇⅡ）诱导血管平滑肌细胞（ＶＳＭＣｓ）内丝裂原活化蛋白激酶（ＭＡＰＫ）激活中的作用,以了解ＡｎｇⅡ促ＶＳＭＣｓ增殖的信号转导过程。将合成的反义ｃ－ｓｒｃ寡脱氧核苷酸（ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅ－ｏｔｉｄｅｓ,ＯＤＮｓ）以脂质体包裹转染培养的大鼠ＶＳＭＣｓ,用Ｗｅｓｔｅｒｎ印迹测得细胞裂解液中ｐｐ６０ｃ－ｓｒｃ含量明显下降,免疫沉淀方法测得ｐｐ６０ｃ－ｓ     

The synergistic induction of cyclooxygenase-2 in lung fibroblasts by angiotensin II and pro-inflammatory cytokines

Although we have demonstrated that Angiotensin II (Ang II) signaling plays a role in colon and lung tumorigenesis, the precise mechanisms by which Ang II stimulates tumorigenesis remain unclear. The aim of this study was to investigate the synergistic induction of COX-2 by Ang II and pro-inflammatory cytokines in lung fibroblasts. We also compared the efficiencies of Ang II-dependent COX-2 induction in lung epithelial cells and stromal cells. Ang II induced COX-2 expression in lung fibroblasts in a dose-dependent manner (10(-9) to 10(-7) M) through the Ang II subtype 1 receptor (AT(1)). In addition, Ang II synergistically stimulated the induction of COX-2 by pro-inflammatory cytokines, IL-1beta, or TNF-alpha. Our results indicate that the pro-tumorigenic function of Ang II is attributable, in part, to its strong stimulatory effect of COX-2 expression in lung fibroblasts in which synergistic stimulation with pro-inflammatory cytokines was evident. It is also suggested that the AT(1) receptor in lung fibroblasts may be a rational target for chemoprevention of lung cancer.     

Intraluteal release of angiotensin II and progesterone in vivo during corpora lutea development in the cow: effect of vasoactive peptides.

The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.     

Localization of angiotensin II in pig ovary and its effects on oocyte maturation in vitro

The renin-angiotensin system (RAS) has been found in mammalian ovarian tissue; however, its physiological role is unclear. This study examined the content of angiotensin II (Ang II) in porcine follicular fluid (pFF), Ang II localization and its receptors in ovary, and the effects of Ang II on porcine oocyte maturation. The concentrations of Ang II were 6951.82 +/- 1295.83, 3502.99 +/- 679.10, 3147.89 +/- 690.60, and 2545.92 +/- 407.01 pg/ml in pFF from small, medium, large, and extra-large follicles, respectively. In addition, Ang II was found on zona pellucidae (ZP) and granulosa cells by immunoreactive staining. The distribution of AT1, an Ang II receptor subtype, was in accordance with that of Ang II. However, AT2, another Ang II receptor, was mainly distributed in the stroma and thecal layers of follicles. When oocytes were cultured in media containing various concentrations of Ang II, a higher (P<0.05) proportion of oocytes reached metaphase II (MII) in the medium with 100 ng/ml (87.0%) than without Ang II (61%). When oocytes from different sizes of follicles were separately cultured in media containing 100 ng/ml Ang II, maturation rates were significantly higher in oocytes from small (61.5%) and medium (85.1%) follicles than that of their controls (45.1 and 72.6%, respectively). However, addition of Ang II inhibited nuclear maturation in oocytes from large follicles (77.8% versus 87.3%). Fertilization and male pronuclear (MPN) formation rates of oocytes matured in medium containing 100 or 1000 ng/ml of Ang II were higher (P<0.05) than that of oocytes matured in medium containing 0 or 10 ng/ml Ang II. Glutathione content in oocytes cultured for 44 h in medium containing 100 or 1000 ng/ml of Ang II was also higher (P<0.01) than that of oocytes cultured in medium containing 0 or 10 ng/ml Ang II. In conclusion, Ang II was present in porcine ovaries and may regulate follicle growth and oocyte maturation.