Combinations of palytoxin or 12-O-tetradecanoylphorbol-13-acetate and recombinant human insulin growth factor-I or insulin synergistically stimulate prostaglandin production in cultured rat liver cells and squirrel monkey aorta smooth muscle cells

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) or the non-TPA-type tumor promoter, palytoxin, recombinant human insulin growth factor-I (IGF-I) and insulin synergistically stimulate prostaglandin production in rat liver cells (the C-9 cell line). Combinations of palytoxin or TPA with recombinant human IGF-I or insulin also synergistically stimulate deesterification of cellular lipids in C-9 cells prelabelled with [3H]arachidonic acid. With both types of stimulations, prostaglandin production or deesterification, the synergistic response of the IGF-I and insulin is greater with palytoxin than with TPA. Production of prostaglandin E2 and F2 alpha by squirrel monkey smooth muscle cells incubated in the presence of TPA and insulin also is greater than the sum of the two effects taken independently.     

Effect of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI₂ production (measured as 6-keto-PGF_1α in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI₂ production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca²⁺-ionophore a-23187, but not the PGI₂ synthesis stimulated by exogeneous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

LONGTMR3IGF-I as a more potent alternative to insulin in serum-free culture of HEK293 cells

LONG^TMR³IGF-I, an analogue of insulin-like growth factor (IGF)-I, was specifically engineered for use in biopharmaceutical protein production in mammalian cells. LONG^TMR³IGF-I is capable of supporting the growth and survival of Chinese hamster ovary cells in serum-free media at concentrations at least 200-fold lower than required for insulin. LONG^TMR³IGF-I also acts as a more potent growth and survival factor than either insulin or native IGF-I in SF culture of human embryonic kidney (HEK293) cells. To investigate the basis of the enhanced potency of LONG^TMR³IGF-I we have examined the mechanism of action of these mitogens in HEK293 cells. All mitogens tested were found to activate the TypeI IGF receptor (IGF-IR) and insulin receptor (IR) in a dose-responsive manner. However, the level of activation of both receptors after stimulation with LONG^TMR³IGF-I, at lower concentrations, was greater than with either insulin or IGF-I. The greater potency of LONG^TMR³IGF-I in activating the IR, despite having a low affinity for IRs, suggests the presence of heterotetrameric IGF-IR/IR dimers. Interestingly, the decrease in IGF-IR activation at higher concentrations of LONG^TMR³IGF-I suggests that the dose-response curve may be bell-shaped.     

Regulation of prostaglandin synthesis by thyrotropin, insulin or insulin-like growth factor-I, and serum in FRTL-5 rat thyroid cells.

The present report shows that thyrotropin (TSH) regulates all three steps involved in prostaglandin synthesis in FRTL-5 rat thyroid cells, i.e. arachidonic acid release from membrane phospholipids, cyclooxygenase (prostaglandin H synthase) action, and individual prostaglandin formation; however, its action at specific steps may require the presence of, or can be duplicated by, insulin, insulin-like growth factor-I (IGF-I), and/or a serum factor. Thus, TSH releases free arachidonic acid from rat FRTL-5 thyroid cells whose phospholipid fraction is radiolabeled with [3H]arachidonic acid; this action involves a pertussis toxin-sensitive G protein, is not cAMP mediated, and does not require insulin or 5% serum. To quantitate TSH effects on cyclooxygenase activity and on individual prostaglandin formation, a homogenate system and a rapid reversed-phase high pressure liquid chromatography procedure have been developed to measure cyclooxygenase metabolites. TSH increased cyclooxygenase activity in homogenates only if the cells were also exposed to insulin, IGF-I, and/or 5% calf serum; TSH alone had no apparent effect on the activity. Maximal activation, 4-fold over basal/micrograms of DNA, took 36 h to achieve and reflected, at least in part, an increase in cyclooxygenase gene expression. Like cyclooxygenase activity, induction of prostaglandin E2 production required 2 or more factors, i.e. TSH plus insulin/IGF-I or TSH plus insulin/IGF-I plus serum. Increased production of prostaglandin D2, could, however, be detected if cells were treated with TSH alone and the TSH activity could be duplicated by insulin, IGF-I, or calf serum alone.     

Actions of vanadate of arachidonic acid metabolism by cells in culture

Sodium vanadate (11 μM) amplified the PGI₂ production of rat liver cells (the C-9 cell line) incubated with thrombin, platelet activating factor, lysine-vasopressin, the Ca²⁺-ionophore A-23187, interleukin-1ß, 12-tetradecanoylphorbol-13-acetate, teleocidin, epidermal growth factor, palytoxin, thapsigargin and colchicine but not that stimulated by exogenous arachidonic acid. Sodium vanadate (2.2 μM) also amplified PGF_2α production of dog kidney cells (the MDCK cell line) incubated with norepinephrine and, at 0.4 μM, PGI₂ production of bovine aorta smooth muscle cells stimulated by serotonin. Sodium vanadate (55 μM) did not affect production of PGE₂ and PGF_2α in rat basophil leukemia cells (the RBL-1 cell line) stimulated by the Ca²⁺-ionophore A-23187, but did inhibit synthesis of peptide-containing leukotrienes and 12-hydroxyeicosatetraenoic acid. When used with cultured cells at micromolar concentrations, vanadate is known to inhibit protein tyrosine-phosphate phosphatases. These results suggest that in some cells deesterification of lipids is positively regulated, at least in part, by phosphorylation of tyrosine whereas in leukocytes, lipoxygenase activities are negatively regulated, at least in part, by phosphorylation of tyrosine.     

Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC₃H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE₁]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC₃H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC₃H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC₃H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC₃H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation. J. Cell. Physiol. 174:293–300, 1998. © 1998 Wiley-Liss, Inc.     

Amplification of prostaglandin I2 production by thapsigargin.

Rat liver cells (the C-9 cell line) are synergistically stimulated to produce prostaglandin I2 (PGI2) when incubated in the presence of thapsigargin and several recombinant human growth factors or tumor promoters, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and aplysiatoxin, but not acidic fibroblast growth factor (aFGF). The production of PGI2 by exogenous arachidonic acid in the presence of thapsigargin is not amplified. The effects of varying levels of bFGF on PGI2 production in the presence of thapsigargin are biphasic: at low levels (0.05 nM) bFGF's effect is synergistic; at high levels (24 nM) it is not.     

Prostacyclin production after treatment of rat liver cells with tosyl-L-phenylalanine chloromethyl ketone and 12-O-tetradecanoylphorbol-13-acetate is mediated by a phorbol-ester-stimulated intermediate

The prostacyclin (PGI₂) production in rat liver cells by treatment by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is not dependent upon the simultaneous presence of both ligands. Preincubation of the rat liver cells with TPA followed by addition of TPCK, as well as preincubation of the cells with TPCK followed by addition of TPA, results in PGI₂ production. Maximum production is found after a 10 min incubation with TPA or after a 120 min incubation with TPCK. Preincubation with TPA for longer than 10 min or preincubation with TPCK for longer than 2 h results in reduced stimulation of PGI₂ production. Dexamethasone does not eliminate the effects of either preincubation or simultaneous addition of TPCK and TPA. EGTA does not affect either preincubation reaction but does completely inhibit PGI₂ production after simultaneous addition of the agents. Preincubation of the cells for 30 min with aspirin completely inhibits the TPCK-TPA-stimulated PGI₂ synthesis. The PGI₂ production following exogenous addition of arachidonic acid to the cells is unaffected by prior treatment of the cells with TPA, TPCK, or TPA plus TPCK. Taken together the data suggest that TPA stimulates the production of an intermediate which activates a Ca²⁺-dependent phospholipase activity. The intermediate is inactivated by a protease which is inhibited by the SH-reactive agent TPCK. The released arachidonic acid is oxygenated by the constitutively expressed cyclooxygenase (prostaglandin H synthase-1).     

Differential effects of follicle-stimulating hormone, insulin, and insulin-like growth factor I on hexose uptake and lactate production by rat Sertoli cells

The stimulatory effects of follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor I (IGF-I) on lactate production and hexose uptake by Sertoli cells from immature rats were studied. The time-courses and the maximal stimulatory effects of FSH, insulin, and IGF-I on lactate production were virtually identical. When Sertoli cells were incubated in the presence of FSH in combination with insulin or IGF-I (submaximal doses), additive but no pronounced synergistic effects were observed. The stimulatory effects of FSH and insulin were not dependent on the presence of extracellular calcium. 2-Deoxy-D-glucose (2-DOG), an analogue of D-glucose, was used to investigate the hexose transport system of Sertoli cells. Uptake of 2-DOG was linear in time and virtually all of the intracellular 2-DOG was phosphorylated up to 30 min of incubation; 2-DOG uptake was inhibited by cytochalasin B, but not by cytochalasin E. D-glucose, but not D-galactose, appeared to be an effective competitor of 2-DOG uptake. The Km of 2-DOG uptake was not influenced by FSH, insulin, and IGF-I. FSH had no effect on the Vmax of 2-DOG uptake, whereas insulin and IGF-I caused a 30% stimulation of the Vmax. It is concluded that FSH, insulin, and IGF-I stimulate lactate production by cultured Sertoli cells, but that only insulin and IGF-I stimulate hexose transport. The insulin-like effect of FSH on Sertoli cells may principally involve stimulation of glycolytic enzyme activities.     

Stimulation of proliferation of HL60 cells by low concentrations of 12-O-tetradecanoylphorbol-13-acetate and its relationship to the mitogenic effects of insulin.

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth and differentiation of cultured human acute promyelocytic leukemia (HL60) cells have been studied using cells growing in a fully defined medium consisting of RPMI 1640 supplemented with selenium dioxide, insulin, and either transferrin or ferric citrate. High concentrations of TPA (greater than 1 nM) cause the expected inhibition of proliferation and induction of macrophage-like differentiation. In contrast, in cells deprived of insulin, which continue to grow at a slow rate, lower concentrations of TPA stimulate proliferation without inducing differentiation. A TPA concentration between 0.03 and 0.3 nM will approximately double the long-term rate of thymidine incorporation into DNA and the rate of increase in cell density. Low-TPA becomes progressively less able to stimulate further proliferation as the insulin concentration is increased and is virtually without effect on cells stimulated by an optimal insulin concentration (5 micrograms ml-1). Insulin itself stimulates proliferation to a greater extent than low-TPA, increasing the long-term rate of thymidine incorporation and the rate of increase in cell density by three- to fourfold. The ability of higher concentrations of TPA to induce differentiation is independent of the presence of insulin. Low-TPA also stimulates the short-term incorporation of thymidine (during a 1-h pulse after 1 or 2 days incubation) by three- to fourfold, as compared to a sevenfold stimulation by insulin. The proliferation response to low TPA concentrations provides a useful model for dissecting the signalling pathways that control cell proliferation following stimulation by insulin and activators of protein kinase C.     

Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture

• 1.1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture.
• 2.2. Specific ¹²⁵I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, K_d~0.3nM. Insulin was nearly 100-fold more potent than IGF-I in displacing ¹²⁵I-labelled insulin.
• 3.3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of ¹²⁵I-labelled insulin and ¹²⁵I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types.
• 4.4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.

     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E₂ is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E₂ by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E₂ than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E₂; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E₂ biosynthesis in this tissue.     

A role for somatomedin C as a differentiating hormone and amplifier of hormone action on ovarian cells: studies with synthetically pure human somatomedin C and swine granulosa cells

Isolated swine granulosa cells incubated in chemically defined medium in vitro responded to synthetically pure human somatomedin C/IGF-I in a dose and time-dependent fashion with increased pregnenolone, progesterone and estradiol biosynthesis. These stimulatory actions were not mimicked by growth hormone, proinsulin, desoctapeptide insulin, epidermal growth factor, or fibroblast growth factor. Moreover, somatomedin C/IGF-I augmented the steroidogenic response of granulosa cells to exogenously supplied sterol substrate in the form of low-density lipoprotein, and amplified the stimulatory actions of the classical ovarian effector hormones, estradiol and follicle-stimulating hormone, in a synergistic fashion. The ability of somatomedin C/IGF-I to stimulate estradiol production on the one hand, and to act synergistically with estradiol to stimulate progesterone biosynthesis on the other hand, suggests a unique intrafollicular mechanism for amplifying progestin biosynthetic capacity in granulosa cells.     

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.     

The stimulations of arachidonic acid metabolism by recombinant murine interleukin 1 and tumor promoters or 1-oleoyl-2-acetyl-glycerol are synergistic

Recombinant murine IL 1 stimulated arachidonic acid metabolism by rat liver cells (the C-9 cell line) and squirrel monkey smooth muscle cells, and in the presence of tumor promoters this stimulation was synergistic. In the rat liver cells that had been prelabeled with [3H]arachidonic acid, the release of 6-keto-PGF1 alpha and arachidonic acid also was stimulated by the IL 1, and this release was synergistic in the presence of TPA. 1-Oleoyl-2-acetyl-glycerol (OAG) stimulated prostaglandin production, and IL 1 synergized the prostaglandin production in the presence of OAG. OAG and TPA mimic the endogenous activator of protein kinase C, 1,2-diacylglycerol, and therefore IL 1 may amplify arachidonic acid metabolism during signal transmission processes.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Synergistic inhibition of metabolic cooperation by oleic acid or 12-0-tetradecanoylphorbol-13-acetate and dichlorodiphenyltrichlorethane (DDT) in Chinese hamster V79 cells: Implication of a role for protein kinase C in the regulation of gap junctional intercellular communication

The effects of TPA and/or DDT and oleic acid and/or DDT on gap junction-mediated intercellular communication (i.e. metabolic cooperation) between Chinese hamster V79 cells was examined. Addition of TPA, DDT or oleic acid alone to cocultures of 6t-hioguanine-resistant (6-TG ^R ) and 6-thioguanine-sensitive (6-TG ^S ) V79 cells significantly increased the recovery of 6-TG ^R cells indicating inhibition of metabolic cooperation. In the presence of TPA and DDT or oleic acid and DDT the observed recovery of 6-TG ^R cells was significantly greater than the expected (calculated) additive 6-TG ^R cell recovery. No synergistic increases in 6-TG ^R cell recovery were observed when co-cultures of V79 cells were exposed to dieldrin and DDT. These results indicate that TPA and DDT or oleic acid and DDT can act synergistically to inhibit metabolic cooperation. These data suggest a role for protein kinase C in the regulation of gap junction-mediated intercellular communication.Abbreviations DDT dichlorodiphenyltrichlorethane - MC metabolic cooperation defective - 6-TG 6thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate     

Effect of insulin-like growth factor I and its interaction with gonadotropins on in vitro maturation and embryonic development,cell proliferation,and biosynthetic activity of cumulus-oocyte complexes and granulosa cells in buffalo

In this study we have examined the effect of insulin like growth factor I (IGF-I) and its interaction with gonadotropins in the presence or absence of granulosa cell coculture on in vitro oocyte maturation (IVM) and their subsequent embryonic development in buffalo. We also have examined the role of IGF-I alone or in combination with gonadotropins on DNA synthesis, steroidogenesis, and protein synthesis of cumulus-oocytes complexes (COCs) and granulosa cells. Results showed that IGF-I stimulates oocytes maturation in a dose-dependent manner, with maximal effect at a dose of 100 ng/ml (P < 0.05). IGF-I showed positive interaction with follicle-stimulating hormone (FSH) in the presence or absence of granulosa cells on meiotic maturation and synergistically enhanced DNA synthesis, protein synthesis, and steroidogenesis in the presence of granulosa cells. This synergistic effect is mainly caused by the increase of IGF-I receptors in granulosa cells by FSH, as evident by [¹²⁵I]IGF-I binding study. Luteinizing hormone (LH), however, was found to suppress IGF-I and IGF-I + FSH stimulated oocyte maturation. Addition of LH to cultures containing IGF-I + FSH, on the contrary, caused a significant increase in oocyte maturation when cocultured with granulosa cells. Addition of IGF-I during IVM significantly improve cleavage and blastocyst development rate over the control group. However, there was no cumulative effect when IGF-I and gonadotropins were present together. Addition of granulosa cells during IVM, however, enhanced blastocyst development in the IGF-I + FSH and IGF-I + FSH + LH groups. Our results demonstrated that IGF-I is a major follicular factor responsible for stimulating oocyte maturation in the buffalo. Interaction between IGF-I and FSH suggests that they seem to act synergistically as an autocrine and paracrine regulator of granulosa cells and therefore together promote mitosis, steroidogenesis, and protein synthesis. Mol. Reprod. Dev. 49:277–285, 1998. © 1998 Wiley-Liss, Inc.     

Oxysterol activation of arachidonic acid release and protaglindin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase activity

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.