Case of syndrome of headache with neurological deficits and cerebrospinal fluid lymphocytosis (HaNDL) with focal slowing on electroencephalogram.

We describe a case of headache and neurological deficits with cerebrospinal fluid (CSF) lymphocytosis in a patient presenting with a 3-week history of recurrent severe headaches associated with negative sensory symptoms and dysphasia. The patient had no cardiovascular risk factors and no family history of migraines. Neurological examination was unremarkable. Cerebral magnetic resonance imaging was unremarkable. CSF analysis revealed lymphocytosis (leucocytes 84 × 10(6)/L, 100% lymphocytes). Extensive laboratory investigations of CSF and serum did not reveal an infectious, autoimmune or metabolic cause. Visual evoked potentials were normal. Awake electroencephalogram revealed intermittent 3-5 Hz generalised slowing and frontal intermittent rhythmic delta activity, without epileptiform discharges. Repeat CSF analysis showed marked reduction of the total leucocyte count and remained negative for infectious aetiology. Propranolol was commenced, and no recurrence of headache or neurological symptoms was observed at follow-up. An extensive literature review on the topic is discussed.

Reversible posterior leukoencephalopathy syndrome: diagnosis and management in the setting of lung transplantation.

Reversible posterior leukoencephalopathy syndrome (RPLS) is a potentially devastating early complication of calcineurin inhibitor (CNI) therapy in solid organ transplantation. Management centres on cessation of CNI therapy; however, this strategy is complicated in lung transplantation because of the threat of allograft rejection, or, if CNI is replaced with mammalian target of rapamycin-based immunosuppression, poor wound healing and bronchial dehiscence. We describe four cases of RPLS after lung transplantation, emphasizing the diagnostic and management approach required to maintain a healthy allograft and ensure that RPLS is, as the name suggests, reversible.

Low molecular mass polypeptide-2 in human trophoblast: over-expression in hydatidiform moles and possible role in trophoblast cell invasion.

Embryo implantation involves invasion of placental extravillous trophoblast cell (EVTs) into the uterus. Hyperactive EVT invasion occurs in hydatidiform moles and choriocarcinomas. We have previously demonstrated that the 20S proteasome is involved in mouse embryo implantation and its action is mediated via regulating the expression and activities of matrix metalloproteinase (MMP)-2 and MMP-9 in the EVTs. Our objective was to investigate whether low molecular mass polypeptide-2 (LMP2), a beta subunit of the 20S proteasome, is involved in the regulation of human trophoblast invasion. Normal human placentas or placentas from hydatidiform mole patients were collected and the expression of LMP2 in different cell types including trophoblastic column (TC), cytotrophoblast cells (CTB) and syncytiotrophoblast (STB) under different pathological states were studied by immunohistochemical analysis. Furthermore, the effect of LMP2 or proteasome on cell invasion was measured by using RNAi and inhibitors in a Matrigel invasion assay system in HTR-8/SVneo cells, a human invasive extravillous trophoblast cell line. Changes in the invasion-related molecules including MMP-2 and MMP-9 were also examined by using real time PCR and gelatin zymography. We demonstrated that the expression of LMP2 in TC of partial hydatidiform mole and complete hydatidiform mole, is higher than that in TC of normal human placentas. Besides, LMP2 knockdown significantly attenuated IL-1beta-induced cell invasion in vitro, a response readily induced by proteasome inhibitors. In summary, over-expression of the 20S proteasome beta-subunit LMP2 in trophoblast cells of hydatidiform moles may contribute to its highly invasive phenotype.

Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance.

The present study determines if (1) basal protein levels of nitric oxide (NO) synthases (eNOS, iNOS, and nNOS) are different in cisplatin-sensitive (OV2008) and counterpart cisplatin-resistant (C13(*)) human ovarian cancer cells, (2) cisplatin alters NOS levels, (3) NO donor causes apoptosis and p53 upregulation, (4) NO donor sensitizes C13(*) cells to cisplatin via p53 upregulation (determined by p53 siRNA gene-knockdown), and (5) inhibition of endogenous NOS alters cisplatin-induced apoptosis. Basal iNOS levels were higher in OV2008 cells than in C13(*) cells. Cisplatin upregulated iNOS, but dramatically reduced eNOS and nNOS, in OV2008 cells only. Failure of cisplatin to upregulate iNOS and downregulate eNOS/nNOS in cisplatin-resistant C13(*) cells may be an aetiological factor in the development of resistance. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased p53 protein levels and induced apoptosis in both cell types, and enhanced cisplatin-induced apoptosis in C13(*) cells in a p53-dependent manner (i.e., enhancement blocked by p53 siRNA). Specific iNOS inhibitor 1400W partially blocked cisplatin-induced apoptosis in OV2008 cells. In cisplatin-resistant C13(*) cells, blocking all NOSs with N(G)-amino-L-arginine dramatically changed these cells from cisplatin-resistant to cisplatin-sensitive, greatly potentiating cisplatin-induced apoptosis. The data suggest important roles for the three NOSs in regulating chemoresistance to cisplatin in ovarian cancer cells.

Regulation of p53 and suppression of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian cancer cells.

Dysregulated apoptosis plays a critical role in the development of a number of aberrant cellular processes, including tumorigenesis and chemoresistance. However, the mechanisms that govern the normal apoptotic program are not completely understood. Soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP) promote mammalian cell viability via an unknown mechanism and p53 status is a key determinant of cell fate in human ovarian cancer cells. Whether an interaction exists between these two determinants of cell fate is unknown. We hypothesized that basal sGC activity reduces p53 content and attenuates p53-dependent apoptosis in human ovarian cancer cells. Suppression of sGC activity with the specific inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) lowered cGMP content, and increased p53 protein content and induced apoptosis in three ovarian cancer cell lines, effects which were attenuated by the cGMP analog 8-Br-cGMP and by Atrial Natriuretic Factor, an activator of particulate guanylyl cyclase, which circumvent the inhibition of sGC. ODQ prolonged p53 half-life, induced phosphorylation of p53 on Ser15, and upregulated the p53-dependent gene products p21, murine double minute-2, and the proapoptotic, p53-responsive gene product Bax. ODQ activated caspase-3, and ODQ-induced apoptosis was inhibited by overexpression of X-linked inhibitor of apoptosis Protein. Pretreatment with the specific p53 inhibitor pifithrin or downregulation of p53 using a specific small inhibitory RNA significantly attenuated ODQ-induced apoptosis. Moreover, ODQ-induced upregulation of p21 and Bax and ODQ-induced apoptosis were significantly reduced in a p53 mutant cell line relative to the wild-type parental cell line. Thus, the current study establishes that basal sGC/cGMP activity regulates p53 protein stability, content, and function, possibly by altering p53 phosphorylation and stabilization, and promotes cell survival in part through regulation of caspase-3 and p53.

DLX1 acts as a crucial target of FOXM1 to promote ovarian cancer aggressiveness by enhancing TGF-ß/SMAD4 signaling.

Recent evidence from a comprehensive genome analysis and functional studies have revealed that FOXM1 is a crucial metastatic regulator that drives cancer progression. However, the regulatory mechanism by which FOXM1 exerts its metastatic functions in cancer cells remains obscure. Here, we report that DLX1 acts as a FOXM1 downstream target, exerting pro-metastatic function in ovarian cancers. Both FOXM1 isoforms (FOXM1B or FOXM1C) could transcriptionally upregulate DLX1 through two conserved binding sites, located at +61 to +69bp downstream (TFBS1) and -675 to -667bp upstream (TFBS2) of the DLX1 promoter, respectively. This regulation was further accentuated by the significant correlation between the nuclear expression of FOXM1 and DLX1 in high-grade serous ovarian cancers. Functionally, the ectopic expression of DLX1 promoted ovarian cancer cell growth, cell migration/invasion and intraperitoneal dissemination of ovarian cancer in mice, whereas small interfering RNA-mediated DLX1 knockdown in FOXM1-overexpressing ovarian cancer cells abrogated these oncogenic capacities. In contrast, depletion of FOXM1 by shRNAi only partially attenuated tumor growth and exerted almost no effect on cell migration/invasion and the intraperitoneal dissemination of DLX1-overexpressing ovarian cancer cells. Furthermore, the mechanistic studies showed that DLX1 positively modulates transforming growth factor-ß (TGF-ß) signaling by upregulating PAI-1 and JUNB through direct interaction with SMAD4 in the nucleus upon TGF-ß1 induction. Taken together, these data strongly suggest that DLX1 has a pivotal role in FOXM1 signaling to promote cancer aggressiveness through intensifying TGF-ß/SMAD4 signaling in high-grade serous ovarian cancer cells.

Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells.

Cisplatin-centered chemotherapy is a key treatment for ovarian cancer, but resistance to chemotherapeutic agents remains a major cause of treatment failure. Multiple factors are known to contribute to the development of this chemoresistance. Although it has been demonstrated that X-linked inhibitor of apoptosis protein (Xiap) prevents apoptosis by inhibiting effector caspases, if and how it is important in chemoresistance in ovarian cancer has not been studied. The effects of Xiap down-regulation and/or restoration of wild type p53 by recombinant adenovirus infection were examined on four ovarian epithelial cancer cell lines [C13*, A2780-s (wild type p53), A2780-cp (mutant p53), and SKOV3 (null p53)]. Apoptosis and protein expression (e.g., Xiap, caspase-3, p53, MDM2, and p21waf1) were assessed by Hoechst 33258 stain and Western blot, respectively. We demonstrated that Xiap down-regulation following adenoviral antisense expression induces apoptosis in the wild-type p53 cells, but not in the mutated or null cells. Xiap down-regulation resulted in caspase-3 activation, caspase-mediated MDM2 processing, and p53 accumulation. Restoration of wild type p53 in the p53-mutated or -null cells significantly enhanced the proapoptotic effect of Xiap antisense expression. Down-regulation of Xiap induced apoptosis in chemoresistant ovarian cancer cells, a process dependent on p53 status.

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells.

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.

Comparisons of brain, uterus, and liver mRNA expression for cytochrome p450s, DNA methyltransferase-1, and catechol-o-methyltransferase in prepubertal female Sprague-Dawley rats exposed to a mixture of aryl hydrocarbon receptor agonists.

Non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants that exert their toxicity mostly through activation of the aryl-hydrocarbon receptor (AhR), and are referred to as AhR agonists. The objective was to study, by real time reverse-transcriptase-polymerase chain reaction (RT-PCR), the effects of postnatal exposure to a reconstituted mixture of AhR agonists present in breast milk (3 non-ortho PCBs, 6 PCDDs, and 7 PCDFs, referred to here-in-after as AhRM) on mRNA expression of estrogen receptor (ERalpha), enzymes involved with the metabolism of estrogens [catechol-o-methyltransferase (Comt), cytochrome P450 (Cyp)1A1, 1B1 and 2B1], and DNA methyltransferase-1 (Dnmt1), in brain areas, liver and uterus of immature female rats. Neonates were exposed by gavage during postnatal day (PND) 1-20 with dosages equivalent to 1, 10, 100, and 1000 times the estimated average human exposure level, and were sacrificed at PND 21. None of the end points were affected in uterine cross-sections, or in samples of uterine tissue layers collected by laser capture microdissection. At 1000x, the AhRM reduced Dnmt1 mRNA abundance to 28% and 32% of control in the liver and hypothalamus, respectively. In the brain, Cyp1A1 was increased (409%) but ERalpha was reduced (66%). Similarly, mRNA abundance for Comt isoforms was reduced in the liver (45%) and brain areas (55-70%). AhRM at 100x, the lowest effective dose, exerted a 220% increase in brain cortex Comt [membrane bound (Mb)], a 219% increase in hepatic Cyp1B1, and a 63% decrease in hepatic Comt (soluble (S)+Mb). These results support the possibility that early exposure to environmental contaminants could lead to effects mediated by changes in DNA methylation and/or estrogen metabolism and signaling.

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.

Calcium requirement in the gonadotropic regulation of rat granulosa cell progesterone production.

Granulosa cells from estrogen-treated immature rats were incubated in chemically defined media containing FSH, cholera toxin, (Bu)2AMP, and/or 3-isobutyl-1-methyl-xanthine in the presence and absence of a calcium chelator (EGTA), an inhibitor of uptake of extracellular calcium [verapamil or lanthanum (La)], or an inhibitor of calmodulin [trifluoperazine or 1-[bis-(p-chlorophenyl)methyl]3-[2,4-dichloro-beta-(2, 4-dichlorobenzyloxy)phenethyl]imidazolium chloride]. Regardless of the presence of 3-isobutyl-1-methyl-xanthine, FSH stimulated cAMP and progesterone production. La inhibited the basal and FSH-stimulated synthesis of progesterone and gonadotropin-enhanced cAMP production. Whereas the net synthesis of cAMP was also inhibited by La in the presence of 3-isobutyl-1-methyl-xanthine, it was increased in the absence of the phosphodiesterase inhibitor. EGTA decreased the basal, FSH-stimulated, and cholera toxin-stimulated production of progesterone but not of cAMP. While (Bu)2cAMP stimulated progesterone production, this response was markedly attenuated by La, verapamil and EGTA. Addition of the calmodulin inhibitors to the granulosa cell incubations also markedly decreased the FSH-stimulated production of cAMP and progesterone as well as the steroidogenic response to the dibutyryl cyclic nucleotide. These findings suggest that calcium plays an important role in the regulation of progesterone production in the rat granulosa cell. In addition to its requirement in the control of cellular cAMP levels, calcium may be involved at a step(s) in the steroidogenic pathway distal to the cAMP cascade.

Microtubules and the gonadotropic regulation of granulosa cell steroidogenesis.

The involvement of microtubules in the gonadotropic regulation of granulosa cell steroidogenesis was assessed at the preantral (E2-cells) and antral (PMS-cells) stages of follicular development. The influence of agents that alter microtubule-tubulin equilibrium on basal and FSH-stimulated progesterone production was determined in vitro and compared with that on microtubule integrity and organization using immunofluorescence. Basal and FSH-stimulated progesterone production was approximately 2-fold higher in PMS-cells than in E2 cells. Colchicine and nocodazole, two agents that depolymerize microtubules, significantly stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one production in PMS-cells. Although progesterone production by E2-cells was increased by nocodazole, the amount produced was considerably less than that produced by PMS-cells. FSH-stimulated progesterone biosynthesis was reduced by colchicine and nocodazole in both cell types. Taxol, an agent that stabilizes microtubules, markedly reduced FSH-stimulated progesterone production in both E2- and PMS-cells, but failed to exert a comparable effect on basal steroid production. A close association existed between the concentrations of colchicine, nocodazole, and taxol that altered basal and/or FSH-stimulated steroidogenesis and those that affected microtubule organization and/or distribution. Whereas granulosa cells appeared flattened with numerous cytoplasmic processes after 24 h of culture in medium alone, they were almost spherical and devoid of projections after culture with these agents. FSH-stimulated cells also occupied less area than controls, although cytoplasmic processes were present. These findings indicate an involvement of microtubules in the regulation of granulosa cell steroidogenesis. It is proposed that one of their roles is to facilitate the movement of cholesterol from lipid droplets to mitochondria, possibly by bringing these cellular inclusions closer together.

Interactions of transforming growth factor-alpha and -beta and luteinizing hormone in the regulation of plasminogen activator activity in avian granulosa cells during follicular development.

This study examined the influence of transforming growth factor-alpha (TGF alpha), TGF beta, and LH on progesterone (P4) secretion and plasminogen activator (PA) activity in cultured avian granulosa cells from the first (F1), third (F3), and fifth and sixth (F5-6) preovulatory follicles during a 21-h incubation period. PA activity in the cell (PAc) and the medium (PAm) fractions was measured by fibrinolysis and fibrin overlay methods. P4 was determined by RIA. Basal PAc and PAm activities were highest in cell cultures from the less mature (F5-6) follicles and decreased as follicles matured to the F1 stage of development. PAc activity was greater than PAm activity regardless of the stage of follicular maturation. TGF alpha (0.1-10 ng/ml) increased PA activity in cultures of granulosa cells from F1, F3, and F5-6 follicles in a concentration-dependent manner. TGF alpha-induced PAc and PAm activities were observed by 6 and 15 h of incubation, respectively, and increased rapidly between 15-21 h. LH (100 ng/ml) attenuated TGF alpha-induced PA activity by 15 h in cultures of granulosa cells from F1 and F3, but not F5-6, follicles. Basal PA activities were unaffected by the gonadotropin. TGF beta (2-100 ng/ml) stimulated PAc activity in a dose-dependent manner only in cultures of granulosa cells from F5-6 follicles and significantly enhanced TGF alpha-induced PAc and PAm activities in cell cultures from F3 and F5-6, but not F1, follicles. Basal and growth factor-induced PAc and PAm activities corresponded to a mol wt of about 35 kDa, a value consistent with that of the low mol wt uPA species. TGF alpha and TGF beta, alone or in combination, had no effect on basal P4 secretion at all stages of follicular development. TGF alpha, however, decreased LH-induced P4 secretion in F1 and F3 cultures. These results demonstrate a tightly controlled interaction of TGF alpha, TGF beta, and LH in regulating PA activity and P4 secretion during follicular development in the domestic hen.

Regulation of hen granulosa cell prostaglandin production by transforming growth factors during follicular development: involvement of cyclooxygenase II.

PGs are important physiological regulators of granulosa cell proliferation during ovarian follicular development. The rate-limiting step in the synthesis of PGs from arachidonic acid is catalyzed by cyclooxygenase (COX). Although two isoforms of COX (COX I and COX II) have been identified in the ovary, the nature and physiological significance of their regulation by transforming growth factor (TGF) alpha and TGF beta are unclear. The object of the present studies was to investigate the regulation of hen granulosa cell PG production by TGF alpha and TGF beta and the role of COX II in this process. Granulosa cells from the first (F1) and fifth and sixth (F5-F6) largest hen preovulatory follicles were cultured for up to 21 h in the presence of TGF alpha (0-10 ng/ml) and/or TGF beta (0-20 ng/ml). COX protein and messenger RNA (mRNA) levels were determined by Western blot and Northern analysis, respectively. Granulosa cell PGF and PGE secretion was increased by TGF alpha but suppressed by TGF beta in vitro. The increase in PG secretion was accompanied by an elevation of COX II content, which was dose and time dependent, with maximum response observed within 6-12 h. Maximal increase in COX II mRNA abundance was evident at 4 and 8 h in cells from F1 and F5-F6, respectively. Although TGF alpha-stimulated PG secretion was higher in cells from mature follicle (F1), the magnitude of change in COX II mRNA abundance and protein content was, however, not dependent on follicular maturation. TGF beta significantly suppressed basal and TGF alpha-induced COX II transcript levels. COX I transcript, however, was undetectable irrespective of the presence of TGF alpha, duration of culture or the stage of follicular development. In conclusion, the mitogenic response of granulosa cells to TGF alpha is mediated by changes in PG secretion, which are regulated at the level of COX II. Unlike the control of PG secretion, the regulation of COX II by TGF alpha is independent of follicular maturation. Whereas COX II may be important in the signaling cascade for TGF alpha in the regulation of granulosa cell proliferation, the mechanism(s) of regulation of follicular stage-dependent PG secretion by the growth factor is complex and requires further investigation.

Epidermal growth factor elevates intracellular pH in chicken granulosa cells by activating protein kinase C.

Previous studies from our laboratory have demonstrated that epidermal growth factor (EGF), induces intracellular alkalinization in chicken granulosa cells by activating a sodium-dependent and amiloride-sensitive Na+/H+ antiporter. In the present investigation we have examined the possible involvement of protein kinase C (PKC) in the regulation of intracellular pH (pHi) by EGF in chicken granulosa cells. Intracellular pH in granulosa cells obtained from the two largest preovulatory follicles was determined spectrofluorometrically using the dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein. The resting pHi was 6.81 +/- 0.01 (n = 30) when the extracellular pH and sodium concentration were 7.3 and 144 mM, respectively. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA; 50-400 ng/ml) and 1-oleoyl-2-acetylglycerol (OAG; 1-75 micrograms/ml) mimicked the actions of EGF by inducing a concentration-dependent increase in pHi which reached a maximum of 0.25-0.30 pH units. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester with no tumor promoting activity had no effect on pHi. Cytosolic alkalinization was observed within 10 min of the addition of each agent and increased over the 60-min observation period. Like EGF-induced cytosolic alkalinization, the increases in pHi in response to TPA or OAG were dependent on the presence of sodium concentration and were inhibited by amiloride, an inhibitor of the Na+/H+ antiporter. The effects of EGF, TPA, and OAG were attenuated by the PKC inhibitors 5-isoquinolinylsulfonyl-2-methyl piperazine and trifluoperazine. Down-regulation of granulosa cell PKC by pretreatment with TPA (200 ng/ml) for 2.5 h inhibited EGF-, TPA-, and OAG-induced cytosolic alkalinization. The effects of maximally stimulatory concentrations of EGF and TPA on cytosolic alkalinization were not additive. The increases in pHi induced by TPA and OAG, but not by EGF, were dependent on the presence of extracellular Ca++. These studies suggest that the EGF-induced intracellular alkalinization in chicken granulosa cells involves a PKC-mediated activation of the Na+/H+ antiporter.

X-linked inhibitor of apoptosis protein activates the phosphatidylinositol 3-kinase/Akt pathway in rat granulosa cells during follicular development.

X-linked inhibitor of apoptosis protein (XIAP) in granulosa cells is regulated by gonadotropins during follicular development, although the current understanding of the mechanisms by which XIAP suppressed granulosa cell apoptosis is incomplete. In the present study, we investigated the possible involvement of the phosphatidylinositol 3-kinase (PI 3-K) survival pathway in the regulation of granulosa cell fate. Using a fully characterized in vivo model to study the induction of follicular development and atresia in immature rats, we have demonstrated that gonadotropin treatment increased granulosa cell XIAP and phospho-Akt protein contents and suppressed apoptosis. In addition, gonadotropin withdrawal [equine CG (eCG)-primed rats treated with an anti-eCG antibody] induced granulosa cell apoptosis and significantly decreased ovarian weight. The increased apoptosis was accompanied by marked decreases in XIAP expression and phosphorylation of Akt protein. Infection of granulosa cells from eCG-primed rats with adenoviral sense XIAP [lacZ as a control; multiplicity of infection, 1-5] resulted in XIAP overexpression and increased phospho-Akt content, whereas XIAP antisense expression (multiplicity of infection, 10-40) decreased granulosa cell phospho-Akt level and induced apoptosis. Addition of the specific PI 3-K inhibitor LY294002 to the granulosa cell cultures decreased Akt phosphorylation and induced apoptosis in a dose-dependent manner. Taken together, these results demonstrate for the first time the importance and regulation of the PI 3-K survival pathway by XIAP in the control granulosa cell apoptosis.

Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor alpha-induced apoptosis.

Although X-linked inhibitor of apoptosis protein (Xiap) is an important intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-alpha (TNFalpha) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFalpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 microM), and/or adenoviral Xiap sense or antisense complementary DNA. TNFalpha alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTPbiotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFalpha alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expression. Down-regulation of Xiap expression by antisense complementary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-kappaB activation by N-acetyl-cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFalpha. The latter phenomenon was readily attenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFalpha death signaling pathway in granulosa cells. Its expression is regulated by the TNFalpha via a nuclear factor-kappaB-mediated mechanism.

The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells.

The effects of the muscarinic cholinergic receptor agonist carbachol on intracellular calcium ion concentration ([Ca2+]i) and progesterone production was determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the calcium-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 13). There was a 4- to 8-fold increase in [Ca2+]i in 85% (n = 80) of the cells within 10 sec after the addition of a maximally stimulating concentration (2 mM) of carbachol. The initial [Ca2+]i spike was followed by a sustained, but lower, calcium elevation, with superimposed oscillations which returned to resting level after several minutes. Both phases of the calcium transient were inhibited by pretreating the cells with atropine (1 microM), pirenzepine (2 microM) or 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). The sustained phase of the response with its superimposed oscillations, but not the initial spike, was inhibited by pretreating the cells with the calcium channel blockers lanthanum (1 mM), cobalt (5 mM), or methoxyverapamil (50 microM), or by incubating the cells in calcium-free medium. Nifedipine (0.5-20 microM) did not affect the carbachol-induced calcium transient. 8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (50 microM) blocked the sustained-oscillatory phase of the carbachol-induced [Ca2+]i transient, but did not affect the initial spike when added before carbachol (2 mM). Despite its ability to stimulate [Ca2+]i surges, carbachol (10(-6)-10(-3) M) did not affect basal or LH-stimulated cAMP or progesterone production in 24-h cultures. These studies demonstrate that activation of chicken granulosa cell muscarinic receptors causes a rapid increase in [Ca2+]i through the release of Ca2+ from intracellular stores, followed by a sustained elevation of Ca2+ with superimposed oscillations caused by the influx of extracellular Ca2+. These results also indicate that an increase in [Ca2+]i in chicken granulosa cells does not alone stimulate steroidogenesis, since the carbachol-induced increases in [Ca2+]i were not accompanied by increased progesterone production.

Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia.

In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.

Muscarinic activation inhibits T-type Ca2+ current in hen granulosa cells.

The effect of the muscarinic agonist carbachol on Ca2+ currents in hen granulosa cells isolated from the largest ovarian follicle was studied. The major Ca2+ current observed using the perforated patch technique with a recording solution containing 10 mM Ca2+, but no Na+ or K+, exhibited characteristics typical of T-type Ca2+ current: maximal amplitude at -20 mV, rapid inactivation (half-time of 42 +/- 3 msec at -20 mV), inhibition by 100 microM Ni2+, and insensitivity to the dihydropyridine Ca2+ channel antagonist, nifedipine. In all cell studied, carbachol (0.5 mM) caused an inhibition of this current (elicited by depolarizing pulses from -70 to -20 mV) to an average maximal decrease of 90 +/- 2% below basal values. In some 50% of the cells, the Ca2+ current also partially recovered during the 10-min exposure to the muscarinic agonist. These effects were prevented by the muscarinic antagonist atropine (1 microM). To test whether this inhibition was due to increases in intracellular free Ca2+ concentrations ([Ca2+]i), [Ca2+]i was simultaneously measured in fura-2-loaded cells. For cells incubated in normal solution, [Ca2+]i was 0.15 +/- 0.02 microM, but increased to 0.25 +/- 0.6 microM in cells exposed to the recording solution. Under these conditions, carbachol failed to produce the expected [Ca2+]i transients, but, rather, caused a small decrease (8 +/- 2%) in basal [Ca2+]i attributable to its diminution of Ca2+ current. Thus, the results demonstrated an important muscarinic inhibition of the T-type Ca2+ current not related to [Ca2+]i fluctuations. They indicate, on the other hand, that [Ca2+]i can strongly modulate carbachol-induced mobilization of Ca2+ from the intracellular stores.