Mode of action of prostaglandin F2 alpha in human luteinized granulosa cells: role of protein kinase C

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.     

Osmotic regulation of the Na+/myo-inositol cotransporter and postinduction normalization

BACKGROUND: The mechanisms underlying the vascular effects of propofol are not fully understood. Vasopressin, a potent vasoactive peptide, stimulates the arachidonate cascade and the synthesis of prostacyclin (PGI2; the main metabolite of the cascade in vascular smooth muscle cells). Arachidonic acid (AA) release by phospholipases is the rate-limiting step in the cascade. We investigated the mechanisms underlying vasopressin-induced AA release and the effect of propofol on PGI2 synthesis in a rat aortic smooth muscle cell line: A10 cells. METHODS: In cultured A10 cells pretreated with propofol, the stimulation by vasopressin of AA release and PGI2 synthesis was evaluated by measuring [3H]AA and 6-keto PGF1alpha, respectively, in the culture medium. The effects of propofol on vasopressin-induced activation of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D were evaluated by measuring inositol phosphate formation and choline formation, respectively. RESULTS: A phospholipase C inhibitor and a phosphatidic acid phosphohydrolase inhibitor both attenuated vasopressin-induced AA release and PGI2 synthesis, as did a phospholipase A2 inhibitor. Propofol inhibited vasopressin-induced activation of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D, but this effect of propofol was significant only at supraclinical concentration (0.1 mM). Propofol reduced vasopressin-induced PGI2 synthesis. The inhibitory effect was observed at concentrations (10 microM-0.1 mM) higher than those used clinically. CONCLUSIONS: Propofol suppresses the arachidonate cascade caused by vasopressin at least partly by inhibiting phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D, resulting in the inhibition of PGI2 synthesis. Propofol-mediated inhibition of vasopressin-stimulated synthesis of PGI2 may reduce the vasorelaxation by propofol.     

Inhibition of thrombin-induced contractile responses by protein kinase inhibitors in porcine pulmonary arteries

The clotting enzyme thrombin is known to cause receptor-mediated contractile effects in isolated blood vessels. In the present studies the influence of protein kinase inhibitors on the contractile response of porcine pulmonary arteries to thrombin (3 U/ml) was investigated. Endothelium-denuded rings (2-3 mm) from small arteries were placed in organ baths for isometric tension recording. The vessels were preincubated for 30 min with the inhibitors before inducing contractions. In the presence of the protein kinase C (PKC)-inhibitors staurosporine, BIM I (bisindolyl-maleimide I), chelerythrine and Ro 31-8220 (1 microM each), the contractile responses to the PKC activator phorbol 12,13-dibutyrate (PDBu; 50 nM) were diminished by 70-100%. However, for inhibition of thrombin-induced contractions generally higher concentrations of the inhibitors were required. Only staurosporine at 1 microM inhibited the thrombin effect by about 75%. The tyrosine kinase inhibitor erbstatin (30 microM) did not significantly alter the thrombin effect, whereas genistein at 10 microM caused a significant inhibition of contractile responses to both thrombin and PGF2alpha. At 100 microM genistein also inhibited the contractile effects of PdBu and KCl. These studies suggest that activation of both PKC and non-receptor tyrosine kinases seems to be involved in the signal transduction pathways of thrombin-induced contractile effects in isolated vessels.     

Involvement of protein kinase C in agonist-stimulated goldfish ovulation

The effects of two protein kinase C (PKC) inhibitors, calphostin C and staurosporine, on the in vitro ovulation of goldfish (Carassius auratus) oocytes were investigated. Ovulation was stimulated by prostaglandin (PG) F2 alpha (PGF2 alpha, 2.0 micrograms/ml), by sodium orthovanadate (0.1 mM), by a combination of the phorbol ester phorbol 12-myristate-13-acetate (PMA, 0.1 micrograms/ml) and calcium ionophore A23187 (0.05 micrograms/ml), by thapsigargin (0.2 micrograms/ml), and by elevated pH (8.1). In addition, the effects of these inhibitors on the PKC activity of the goldfish follicle wall was determined by use of a specific peptide substrate phosphorylation assay. At 0.1 microM, staurosporine significantly blocked ovulation induced by all agents. However, at lower (0.01 microM) levels it blocked only PMA/A23187-induced ovulation. In contrast, calphostin significantly blocked only PMA/A23187-induced ovulation, although there was a decrease in pH-induced ovulation at lower calphostin concentrations. Both calphostin and staurosporine blocked follicular PKC activity at levels that were inhibitory to ovulation. In addition, staurosporine significantly blocked PKC activity at levels even lower than those needed to block ovulation. The combined results suggest that orthovanadate, PGF2 alpha, and thapsigargin do not require PKC activation for the induction of ovulation, whereas PMA/A23187 does.     

The autometallographic zinc-sulphide method. A new approach involving in vivo creation of nanometer-sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles

Tumor necrosis factor-alpha (TNF-alpha) influences hormone synthesis of many ovarian cell types and can also exert cytotoxic effects, possibly by increasing the synthesis of prostaglandins. The purpose of the present study was to characterize the mechanism of TNF-alpha-stimulated prostaglandin; F2 alpha (PGF2 alpha) production in cultured bovine luteal cells. Inhibitors of RNA and protein synthesis (actinomycin D and cycloheximide, respectively) completely blocked TNF-alpha-stimulated PGF2 alpha production. The phospholipase A2 inhibitor, aristolochic acid, prevented TNF-alpha-stimulated, but not basal, PGF2 alpha production, whereas the phospholipase C inhibitor, compound 48/80, was without effect. The addition of arachidonic acid to cultures did not overcome the inhibitory effects of cycloheximide or aristolochic acid. In conclusion, TNF-alpha-stimulated prostaglandin production by bovine luteal cells is dependent upon the stimulation of phospholipase A2 through mechanisms which require synthesis of RNA and protein.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific.     

Cross-talk between receptor-mediated phospholipase C-beta and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes

Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10(-8)M), phenylephrine (PHE, 10(-5)M) or Ang II (10(-7)M) resulted in a rapid (0-10 min) activation of PLC-beta to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-6)M) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0-5 min) translocation of PKC- immunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-alpha, -delta and -xi immunoreactivities was observed. PMA caused translocation of PKC-alpha, PKC-epsilon as well as PKC-delta. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10(-4)M) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10(-6)M) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-beta and after a lag-phase PLD, whereas PHE and Ang II activate only PLC-beta. PLC-beta stimulated by ET-1, may cross-talk with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response.     

Comparison of the Ca2+ movement by activation of alpha1-adrenoceptor subtypes in HEK-293 cells

We studied the Ca2+ movement induced by activation of alpha1A-, alpha1B- and alpha1D-adrenoceptor subtypes in transfected HEK-293 cells with the fura-2 probe. All these alpha1-AR subtypes induced both Ca2+ release and Ca2+ entry. The effect on Ca2+ release in alpha1b transfected HEK-293 cells was bigger than that in alpha1a and alpha1d transfected HEK-293 cells, and the effects on Ca2+ entry were the same in alpha1a, alpha1b and alpha1d transfected HEK-293 cells. The Ca2+ entry was inhibited by 1 mM NiSO4, but not by nifedipine. Cyclopiazonic acid (CPA) produced a biphasic Ca2+ signal response in Ca2+ medium, and only induced a transient response in Ca2+-free medium. After depletion of CPA-sensitive Ca2+ pool by 10 microM CPA in Ca2+-free medium, 10 microM adrenaline (Adr) still transiently increased [Ca2+]i in three different alpha1-adrenoceptor subtype transfected HEK-293 cells. However, after depletion of adrenaline-sensitive Ca2+ pool by 10 microM Adr, CPA transiently elevated [Ca2+]i only in alpha1a and alpha1d transfected HEK-293 cells, not in alpha1b transfected HEK-293 cells. U73122, a phospholipase C (PLC) inhibitor, inhibited both Ca2+ release and Ca2+ entry induced by activation of alpha1A alpha1B and alpha1D subtypes in transfected HEK-293 cells. These results suggest that HEK-293 cell line contains two functionally separate intracellular Ca2+ pools, CPA-sensitive and Adr-sensitive pools. Activation of alpha1B-AR stimulates Ca2+ release from both CPA-sensitive and Adr-sensitive Ca2+ pools. Alpha1A and alpha1D subtypes induce Ca2+ release only from Adr-sensitive Ca2+ pool.     

Immunodominant minor histocompatibility antigen peptides recognized by cytolytic T lymphocytes primed by indirect presentation

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.     

Regulation of phospholipase D activity in a human oligodendroglioma cell line (HOG)

Oligodendroglial cells express many specific proteins, such as myelin basic protein (MBP), which are physiologically phosphorylated by protein kinase C (PKC). Diacylglycerols are physiological activators of PKC and can be liberated from phospholipids by the direct receptor-mediated activation of phospholipase C (PL-C) or indirectly via the activation of phospholipase D (PL-D). In a well-characterized human oligodendroglioma (HOG) cell line, PL-C (measured by release of [3H]inositol phosphates) and PL-D (formation of [3H]myristoylated or palmitoylated phosphatidylethanol) were activated by both carbachol (blocked by pirenzepine, suggesting an M1 receptor) and histamine (H1 receptor) but not glutamate, bradykinin, or phenylephrine. PL-C stimulation by carbachol or histamine was completely inhibited by short-term treatment (< 30 min) with phorbol ester (TPA), a PKC activator. In contrast, PL-D activation by either carbachol or histamine was stimulated in additive fashion by TPA, suggesting at least two distinct mechanisms for PL-D activation. Down regulation of PKC by prolonged (24 hr) treatment with TPA reversed the inhibitory effects of TPA on PL-C and the stimulatory effects on PL-D. However, the PKC inhibitors H-7 and galactosylsphingosine did not inhibit the TPA-mediated stimulation of PLD while the less-specific PKC inhibitor, staurosporine, was only partially inhibitory. Preexposure of cells to carbachol, greatly reduced both PL-C and PL-D activation by carbachol, suggesting homologous desensitization. Time-course studies indicated that PL-D activation (10 sec or less) was at least as fast as PL-C activation, and the affinity of carbachol and histamine for the receptor coupled to either phospholipase (EC50 = 5-10 microM) was about the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cosmetic results of breast conserving therapy for breast carcinoma. Treatment results from the Heidelberg Radiation Clinic in the years 1984 to 1992]

We examined the effect of an antioxidant and protein kinase inhibitors on prostaglandin E2 (PGE2) release from Balb/c 3T3 mouse fibroblast cells induced by quinolone phototoxicity. Simultaneous administration of sparfloxacin (SPFX) or lomefloxacin (LFLX) at 12.5 to 100 microM and ultraviolet-A (UVA) irradiation for 10 min markedly elevated PGE2 concentration in the incubation medium, whereas levofloxacin (LVFX) at concentrations up to 100 microM and UVA irradiation did not increase PGE2 concentration. Pretreatment with 100 microM pyrrolidine dithiocarbamate (PDTC), an antioxidant, or 1 microM calphostin C, a selective inhibitor of protein kinase C (PKC), completely inhibited the elevation of PGE2 in the 24-h incubation medium; pretreatment with 10 microM H7, a cyclic nucleotide-dependent protein kinase, and PKC or 1 microM herbimycin A, a tyrosine kinase inhibitor, inhibited the PGE2 elevation by 29 to 39%. Conversely, 25 nM staurosporine significantly augmented the PGE2 elevation by quinolones plus UVA. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) were not detected in the incubation medium of 3T3 cells after quinolone plus UVA, corresponding to the lack of effect of antibodies against IL-1alpha, IL-1beta, and TNFalpha on PGE2 release from 3T3 cells. These results suggest that PGE2 production in 3T3 cells by quinolone phototoxicity is modulated by reactive oxygen species, PKC, and tyrosine kinase, but not by IL-1 or TNFalpha.     

Health of older women

We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCalpha and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. G?-6976, a compound that inhibits PKCalpha and beta specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 microM), a specific activator of PKCalpha, beta, and gamma induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCalpha, but not PKCbeta, from cytosol to the particulate fraction. These results suggest that PKCalpha plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCalpha, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCalpha, which activates cPLA2, which liberates AA for prostaglandin synthesis.     

Flufenamic and tolfenamic acids and lemakalim relax guinea-pig isolated trachea by different mechanisms

The effects of K+ channel inhibitors on the relaxations induced by flufenamic and tolfenamic acids and lemakalim were examined in guinea-pig isolated trachea precontracted with prostaglandin F2alpha (PGF2alpha, 1 microM). Flufenamic and tolfenamic acids (0.1-33 microM) and lemakalim (0.01-33 microM) relaxed guinea-pig trachea in a concentration-dependent manner. Tetraethylammonium (0.5-2 mM), a nonspecific inhibitor of K+ channels, inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Charybdotoxin (ChTX, 33-100 nM), an inhibitor of the large Ca2+-activated K+ channels (BK(Ca)), also inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Glipizide (3.3-33 microM), an inhibitor of the ATP-sensitive K+ channels (K(ATP)) inhibited lemakalim-induced relaxation without affecting those induced by flufenamic and tolfenamic acids. Our results indicate that the relaxations of guinea-pig isolated trachea by flufenamic and tolfenamic acids are due to activation of BK(Ca). The relaxant mechanism of flufenamic and tolfenamic acids thus differs from that of lemakalim, an activator of K(ATP).     

The role of a phosphatidylcholine-specific phospholipase C in the production of diacylglycerol for nitric oxide synthesis in macrophages activated by IFN-gamma and LPS

Murine macrophages activated by interferon (IFN)-gamma and bacterial lipopolysaccharide (LPS) produce large amounts of nitric oxide (NO), which is a critical mediator for a variety of biological functions. The expression of this inducible NO synthase (iNOS) involves a protein kinase C (PKC)-dependent pathway, but the mechanism for the PKC activation in this system is unclear. Through analysis of diacylglycerol (DAG) synthesis and choline metabolism in activated macrophages, direct evidence is provided that NO synthesis involves the activation of an unusual phosphatidylcholine-specific phospholipase C (PC-PLC) and not a phosphatidylinositol-specific phospholipase C (PI-PLC) or phospholipase D (PLD).     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

MAP kinase mediates epidermal growth factor- and phorbol ester-induced prostacyclin formation in cardiomyocytes

We studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in epidermal growth factor (EGF)-induced prostacyclin (PGI2) production in cultured, spontaneously-beating neonatal ventricular rat cardiomyocytes. To this purpose, the effect of EGF on cardiomyocyte MAPK phosphorylation, MAPK activity and PGI2-production were investigated, and compared to those induced by the PKC activator 4 beta phorbol 12-myristate 13-acetate (PMA). Both EGF (0.1 microM) and PMA (0.1 microM) induced the rapid and reversible phosphorylation of 42 KDa-MAPK in ventricular cardiomyocytes, responses that were accompanied by transient increases in MAPK activity (190-230% of control values within 5 min), and two- to three-fold increases in PGI2 formation. The tyrosine kinase inhibitors lavendustin (1 microM) and genistein (10 microM) strongly inhibited EGF-induced MAPK activation and PGI2-formation, but had no effect on PMA-stimulated responses. Experiments with the PKC inhibitor CGP 41251 (1 microM) or with PKC-downregulated cells demonstrated that in contrast to the PMA-stimulated responses, EGF-induced MAPK activation and PGI2-production were PKC-independent processes. Investigating the role of MAPK in EGF- and in PMA-promoted PGI2-formation, we found that the MAPK-inhibitor 6-thioguanine (500 microM), as well as the MAPK-kinase-inhibitor PD98059 (50 microM) abolished both EGF- and PMA-stimulated PGI2-production in cardiomyocytes. Our results indicate that MAPK-activation is at the basis of both growth factor receptor and PKC-dependent eicosanoid-formation in ventricular cardiomyocytes, where EGF-induced prostaglandin-production takes place via a PKC-independent pathway.     

Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells

Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i extrusion.     

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 2. Role of protein kinase C-delta in the regulation of eicosanoid production

PGI2 generation by the vessel wall is an agonist for cyclic-AMP-dependent cholesteryl ester hydrolysis. The process of enhanced PGI2 synthesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-protein-mediated PGI2 synthesis. In the studies reported herein, cells generated PGI2 in response to AlF4-, GTPgammaS, and ATP in a dose-dependent manner. G-protein agonists stimulated eicosanoid production principally by activating phospholipase A2, but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A2 and PLCgamma activities. Galphai subunits mediate G-protein agonist-induced PGI2 synthesis, since ATP- and PDGF-induced PGI2 synthesis was inhibited by pertussis toxin. Although cholesterol enrichment reduced arachidonic acid- and PDGF-induced PGI2 synthesis, cholesterol enrichment enhanced PGI2 release in response to AlF4-, GTPgammaS, and ATP. The enhancement of PGI2 release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits. Since cholesterol enrichment inhibited PDGF and AlF4--induced MAP kinase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J. D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36, 9523-9531] (the major mechanism by which phospholipase A2 is activated), these results suggest that cholesterol enrichment induces other alternative signaling pathways leading to phospholipase A2 activation. A PKC-dependent pathway is described herein that is involved in enhanced eicosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI2 production is inhibited by calphostin, and (2) cholesterol enrichment augments the specific translocation of the delta-isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol ester. These data support the concept that, in cells possessing normal levels of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid synthesis in response to G-protein activation; however, under conditions of high cellular cholesterol levels, augmented G-protein-linked eicosanoid production results from enhanced PKCdelta activity.     

Functional characterization of the ocular prostaglandin f2alpha (PGF2alpha) receptor. Activation by the isoprostane, 12-iso-PGF2alpha

Prostaglandin F2alpha (PGF2alpha) is a product of cyclooxygenase-catalyzed metabolism of arachidonic acid. Recently, PGF2alpha analogs have been hypothesized to reduce intraocular pressure via relaxation of the ciliary muscle. To investigate the molecular basis of PGF2alpha receptor (FP) activation in the eye, we cloned the FP from a human ciliary body (hcb) cDNA library. The open reading frame of the hcb-FP cDNA was identical to the uterine FP cDNA. The hcb-FP appeared to be predominantly membrane-localized, as visualized by an FP-specific peptide antibody, and coupled to inositol phosphate formation when stably expressed in HEK 293 cells. Interestingly, the hcb-FP could also be activated by the F2 isoprostane, 12-iso-PGF2alpha, in addition to its cognate ligand, PGF2alpha. 12-iso-PGF2alpha was less potent (EC50 = 5 microM) than PGF2alpha (EC50 = 10 nM) in generating inositol phosphates via the hcb-FP in HEK 293 cells. Both ligands also stimulated mitogenesis in NIH 3T3 cells. Although 12-iso-PGF2alpha caused a dose-dependent activation of the FP, it failed to activate the recombinant human prostacyclin receptor and caused only minimal activation of the thromboxane receptor isoforms stably expressed in HEK 293 cells. Four additional F2 isoprostanes, 8-iso-PGF2alpha, IPF2alpha-I, IPF2alpha-III, and 9beta,11beta-PGF2, caused trivial, or no, activation of the FP. Consistent with these observations, only PGF2alpha and 12-iso-PGF2alpha caused rapid homologous desensitization of FP and also exhibited cross-desensitization, with PGF2alpha resulting in a maximum of approximately 60% desensitization. The human FP may thus be activated specifically, by the free radical-catalyzed F2 isoprostane, 12-iso-PGF2alpha, in addition to the cyclooxygenase product, PGF2alpha. Incidental receptor activation by isoprostanes may complement the actions of PGF2alpha in clinical syndromes where oxidant stress and augmented prostaglandin biosynthesis coincide.