Addition Of Prostaglandin E2 Research Articles

Regulation of fatty acid biosynthesis as a possible mechanism for the mitoinhibitory effect of fumonisin B1in primary rat hepatocytes

The mitoinhibitory effect of fumonisin B1(FB1) on the mitogenic response of epidermal growth factor (EGF) was investigated in primary hepatocyte cultures with respect to the alterations in the ω6 fatty acid metabolic pathway. Fatty acid analyses of hepatocytes showed that EGF treatment resulted in a significant decrease in the relative levels of 20:4ω6 (arachidonic acid) and an increase in 18:2ω6 (linoleic acid). Supplementation of the hepatocyte cultures with 20:4ω6 in the absence of EGF resulted in an increase in the total ω6 and ω6/ω3 fatty acid ratio. Addition of 20:5ω3 (eicosapentaenoic acid) resulted in an increase of the relative levels of the long chain ω3 fatty acids at the expense of the ω6 fatty acids. When 20:4ω6 and 20:5ω3 was added in the presence of EGF, the mitogenic response of EGF was increased and decreased respectively. When compared to the fatty acid profiles in the absence of EGF, the decreased mitogenic response coincided with a decrease of total ω6 fatty acids and total polyunsaturated fatty acids (PUFA). In addition, the saturated and mono-unsaturated fatty acids increased and the polyunsaturated/saturated (P/S) fatty acid ratio decreased which implied a more rigid membrane structure. Addition of prostaglandin E2(PGE2) and prostaglandin E1(PGE1) stimulated and inhibited the mitogenic response respectively. Ibuprofen, a known cyclooxygenase inhibitor, and FB1inhibited the EGF-induced mitogenic response in a dose- dependent manner. The mitoinhibitory effect of FB1on the EGF response was counteracted by the addition of PGE2. FB1also disrupts the ω6 fatty acid metabolic pathway in primary hepatocytes, resulting in the accumulation of C18:2ω6 in phospatidylcholine and triacylglicerol. The disruption of the ω6 fatty acid metabolic pathway and/or prostaglandin synthesis is likely to be an important event in the mitoinhibitory effect of FB1on growth factor responses.

Paradoxical role of PGE2and cAMP in Actinobacillus actinomycetem-comitants strain Y4-induced lymphocyte proliferation

An immune mechanism has been suggested in the pathogenesis of periodontal disease. Actinobacillus actinomycetemcomitants (Aa) has been implicated as one of the etiological agents that induces the major immune response together with a dense infliltrate of inflammatory cells. But the exact role of these immune cells in periodontal disease has not yet been clarified. In this study the T lymphocyte (TL) proliferative response was evaluated after having being exposed to free cell supernatant (SN) from Aa. Aa SN increased TL proliferation. This mitogenic effect of Aa SN was attenuated by pretreating TL with indomethacin (INDO) or acetylsalicilyc acid (ASA) but not by polymyxin B. The inhibitory effect of INDO on cell proliferation was reversed by the addition of prostaglandin E2(PGE2) to the culture assay. Moreover, when immune cells were exposed to Aa SN they were able to generate PGE2at the same time as intracellular levels of cAMP decreased. Both, PGE2release and decrease accumulation of cAMP in TL were blunted by treated lymphocytes with INDO. In this paper we demonstrate that cell free SN from Aa induces a mitogenic effect on murine lymphocytes. The mechanism involves the host's immunecompetent cells and the release of PGE2and appears not to be induced by capsular-like polysaccharide antigen. Results show a paradoxical mitogenic effect of Aa SN accompanied by increased generation of PGE2and decreased production of cAMP by lymphocytes.

Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO

Bacterial products stimulate macrophage tumoricidal activity through release of tumor necrosis factor (TNF) and nitric oxide (NO). We show here that thioglycollate-elicited macrophages acquire cytotoxic activity when cocultured with Mycoplasma arginini-infected YAC-1 tumor cells and release TNF and NO. Fixed mycoplasma-infected cells, supernatants from infected-cell cultures, or purified heat-killed mycoplasma obtained from cell-free cultures were all able to induce TNF and NO production. Thus, the mycoplasma per se and not a product of infected cells induce the release of these molecules. Addition of prostaglandin E2 (PGE2) to the cocultures, which reduced TNF release, or antibodies to TNF, did not affect macrophage cytotoxicity nor NO release. Inhibition of NO production by L-NAME or aminoguanidine reduced the cytotoxicity, and treatment with a NO donor was toxic to YAC-1 cells. These results indicate that M. arginini activates thioglycollate-elicited murine macrophages for NO and TNF release increasing their cytotoxic activity toward YAC-1 cells and that this activity is dependent on NO but not TNF release.

INDOMETHACIN DOES NOT INFLUENCE ALVEOLAR LIQUID CLEARANCE IN ANESTHETIZED SHEEP OR RATS

Active transport of sodium has been shown to be the predominant mechanism involved in alveolar liquid clearance. One regulatory mechanism involved in the modulation of this transport system is cAMP. Although it was initially thought that cAMP could directly modulate transepithelial Na+ transport, recent data suggest that this cAMP modulation could be secondary to the production of arachnidonic acid metabolites. The purpose of this study was thus to evaluate if prostaglandin products could have an indirect or direct role to play in lung liquid clearance. Addition of 10 5 M salmeterol, known to increase intracellular cAMP, to the instilled fluid in rats stimulated lung liquid clearance. However, addition of indomethacin did not influence the stimulating effect of salmeterol. Furthermore, addition of prostaglandin E2 to the instilled fluid did not stimulate alveolar fluid clearance. In order to determine if this response could be species related, we evaluated if indomethacin could modulate alveolar liquid clearance in sheep. Presence of cAMP and aminophylline stimulated lung liquid clearance in sheep, but indomethacin did not inhibit this response. The present study demonstrates that cyclooxygenase products are not involved in the modulation of basal or stimulated alveolar or lung liquid clearance in sheep or rats.

Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts

Orbital fibroblasts exhibit a phenotype distinct from that of other types of fibroblasts. Addition of prostaglandin E2(PGE2) to culture medium elicits a dramatic change in orbital fibroblast morphology. That response is mediated through the generation of cAMP. Orbital fibroblasts can generate high levels of PGE2through induction by proinflammatory cytokines of prostaglandin endoperoxide H synthase-2 (PGHS-2). Here we compare the influence on fibroblast morphology of exogenous PGE2, forskolin, and 8-br-cAMP to that mediated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblasts treated with any of these test compounds appear under phase-contrast microscopy to exhibit a stellate morphology. When these changes were assessed quantitatively by electric cell–substrate impedance sensing (ECIS), it became evident that 8-br-cAMP, forskolin, and PGE2initiated shape changes within 30 min of addition to the culture medium, while effects of the cytokine were first evident after approximately 3.5 h. Dermal fibroblasts failed to respond to any of these compounds with regard to changes in cellular morphology. Analysis of micromotion, manifested as small impedance fluctuations, revealed that orbital fibroblasts treated with 8-br-cAMP exhibit less motion than did untreated cells. These results suggest that orbital fibroblast shape can be altered by several compounds known to alter intracellular cAMP levels. They demonstrate the utility of ECIS in the assessment of very rapid and dynamic cellular events associated with changes in cell morphology.

Effect of cyclo-oxygenase inhibition on in vitro B-cell function after burn injury.

The role of PGE2 in suppression of B-cell function after burn injury was investigated. Splenocytes from burned or sham-burned mice were isolated 8 days after burn injury and cultured with lipopolysaccharide with or without the addition of prostaglandin E2 (PGE2) or indomethacin (Indo). Anti-peptidoglycan polysaccharide immunoglobulin (Ig)M (specific antibody to a bacterial antigen), total IgM, and total IgG levels in culture supernatant and lymphocyte proliferation were measured. All B-cell functions were significantly suppressed by burn injury. PGE2 suppressed all B-cell functions except for IgG synthesis. Indo restored anti-peptidoglycan polysaccharide IgM to normal levels, but did not have a significant effect on suppressed proliferation and total IgM synthesis. IgG synthesis was increased by PGE2 and inhibited by Indo. Although not all B-cell suppression was accounted for by PGE2, this prostaglandin appeared to be a mechanism responsible for impaired antigen specific antibody response and isotype switching. Successful restoration of specific antibody synthesis to bacterial antigen suggests a potential therapeutic role for a cyclo-oxygenase blocking agent after burn injury.

Sodium channel heterogeneity in the apical membrane of porcine thyroid epithelial cells

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl-. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g = 4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g = 10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na(+)-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na(+)-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.

Effect of the Prostaglandin E2, F2.ALPHA. and Indomethacin on the Incorporation of 3H-Methionine in Rat Blastocysts Development.

The effect of the prostaglandin E2, F2α and indomethacin on the incorporation of 3H-Methionine in rat blastocyst development was studied by the method of liquid scintillation technique. The eggs of intact pregnant rats when they were on the morula, early blastocyst, blastocyst, and late blastocyst stages were collected. Blastocysts of delayed implantation rats were also collected on the day 6 and 8. The rate of implantation of 3H-Methinine in delayed blastocysts was smaller than in normal blastocysts. Addition of prostaglandin E2 or F2α had accelerated the incorporation of 3H-Methionine in blastocysts at preimplantation and during delayed implantation (P<0.05). But addition of indomethacin inhibited the incorporation of 3H-Methionine in blastocyst and delayed implantation on the day 6 and 8 (P<0.05).

Essential fatty acid deficiency impairs the responsiveness of renal pelvic sensory receptors

The role of prostaglandins in renal sensory receptor activation was examined in rats fed an essential fatty acid-deficient (EFAD) diet to cause tissue arachidonate depletion. Littermates fed a standard diet were used as controls. In anesthetized rats, the increases in afferent renal nerve activity due to increasing ureteral pressure 2.5, 5, 7.5, 10, 12.5, and 15 mmHg were significantly reduced by the EFAD diet (P < 0.02): 3 +/- 5, 3 +/- 5, 11 +/- 5, 9 +/- 5, 19 +/- 3, and 17 +/- 5%, respectively, in EFAD rats and 23 +/- 11, 36 +/- 15, 50 +/- 15, 52 +/- 8, 72 +/- 17, and 90 +/- 19%, respectively, in control rats. In EFAD rats, addition of prostaglandin E2 (PGE2) to the renal pelvic perfusate restored the afferent renal nerve activity response to increased ureteral pressure toward that in control rats. PGE2 had no effect in control rats. Also the afferent renal nerve activity responses to renal pelvic perfusion with bradykinin at 4, 20, 100, and 500 micrograms/ml were significantly suppressed by the EFAD diet (P < 0.01): 13 +/- 15, 5 +/- 7, 60 +/- 19, and 63 +/- 20%, respectively, in EFAD rats and 122 +/- 23, 142 +/- 31, 172 +/- 19, and 190 +/- 39%, respectively, in control rats. These results demonstrate an important role for arachidonate metabolites, particularly PGE2, in renal sensory receptor activation. Together with our previous studies showing that indomethacin blocks the afferent renal nerve activity responses to increased ureteral pressure or bradykinin, the present studies provide strong evidence for an essential role of prostaglandins in renal sensory receptor activation.

Stimulation of aged human lung fibroblasts by extracellular ATP via suppression of arachidonate metabolism

The mitogenic effect of extracellular ATP on IMR-90 human fibroblasts subjected to in vitro aging was studied. ATP stimulated DNA synthesis and cell proliferation in young cells as much as epidermal growth factor (EGF) or insulin, while it stimulated aged cells to a much greater extent than seen for any other growth factor tested. When combined with EGF or insulin, ATP restored the greatly reduced mitogenic responsiveness of aged cells nearly to the level noted for young cells. Addition of prostaglandin E2 or other agents that elevate cAMP levels resulted in inhibition of DNA synthesis stimulated by EGF or insulin. Furthermore, the basal release of arachidonic acid and prostaglandin E2 and the endogenous levels of cAMP rose during aging and became much greater than in young cells. All three of these changes were suppressed by extracellular ATP. ATP-dependent suppression of cAMP accumulation was pertussis toxin-sensitive. Protein kinase C down-regulation inhibited arachidonate metabolism and enhanced DNA synthesis stimulated by ATP. These studies suggest that ATP exerts its mitogenic effect, especially on aged IMR-90 cells, at least partially by suppression of arachidonate metabolism.

The contraction of hepatic stellate (Ito) cells stimulated with vasoactive substances. Possible involvement of endothelin 1 and nitric oxide in the regulation of the sinusoidal tonus.

We have studied the contractility of liver sinusoidal stellate (Ito) cells stimulated with endothelin 1, nitric-oxide donors and eicosanoids. Contraction and relaxation of stellate cells were detected by the use of a silicone-rubber method that revealed the traction forces exerted by these cells. Endothelin 1 was a strong elicitor for stellate-cell contraction. 78, 55, 59 and 56% of stellate cells were contracted 2.5, 5, 10 and 20 min, respectively, after exposure to 10 nM endothelin 1. The effect of endothelin 1 was dose dependent and still detectable at an endothelin 1 concentration of 100 pM. Concomitantly, an endothelin-dependent formation of inositol phosphates was apparent; values of InsP, InsP2, and InsP3 were 881 +/- 99%, 1965 +/- 368%, and 791 +/- 120% of control, respectively, 20 min after addition of 10 nM endothelin 1. In addition, endothelin 1 caused a transient increase of [Ca2+]i in stellate cells from a basal value of 121 +/- 9 nM to maximal 1015 +/- 86 nM. These endothelin-1 effects were much stronger than those of the thromboxane-A2 analogue U46619 and of prostaglandin F2 alpha. In contrast, Iloprost, prostaglandin E2, and sodium nitroprusside promoted stellate-cell relaxation; for example, 82, 83 and 71% of stellate cells relaxed 5, 10, and 20 min, respectively, after addition of 500 microM sodium nitroprusside to contacted cells. Prostaglandin E2 and Iloprost led to elevation of cAMP levels in stellate cells from a basal value of 9.2 +/- 0.8 pmol/well to 55.1 +/- 8.0 and 122.2 +/- 12.2 pmol/well 10 min after addition of prostaglandin E2 (5 microM) and Iloprost (5 microM), respectively, in the presence of 3-isobutyl-1-methylxanthine (0.5 mM). However, sodium nitroprusside was a trigger for cGMP accumulation. Intracellular cGMP increased from a basal value of 0.9 +/- 0.07 pmol/well to 13.4 +/- 6.7 pmol/well 10 min after addition of 500 microM sodium nitroprusside into the medium. It is interesting that Iloprost and sodium nitroprusside also induced the disappearance of actin stress fibers in contracted cells; F-actin stress fibers became less numerous and de-aggregated; more than 90% of stellate cells were void of stress fibers after 10 microM Iloprost treatment for 30 min. Thus, endothelin 1, eicosanoids and sodium nitroprusside are able to modulate the contractility of stellate cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin E2 stimulates ion transport in prairie dog gallbladder.

The effects of prostaglandins, and specifically prostaglandin E2, on gallbladder ion transport were examined in the prairie dog. Gallbladders were mounted in an Ussing chamber and baseline short-circuit current, potential difference, and tissue resistance were measured. Addition of arachidonic acid (10(-4) M, mucosal surface) produced sustained elevations in short-circuit current and potential difference (P < 0.05), with mild reductions in resistance. In a second set of tissues, indomethacin exposure (10(-6) M) resulted in a significant (P < 0.02) decrease in short-circuit current and potential difference, with an increase in resistance. Subsequent addition of prostaglandin E2 (10(-7) M, serosal surface) fully reversed these changes and led to a significant increase in short-circuit current and potential difference (P < 0.001) with a return of resistance to baseline values. These findings suggest that endogenous prostaglandins mediate gallbladder ion transport.

The role of CD18 in IL-8 induced dermal and synovial inflammation.

1. The intradermal administration of endothelial IL-8 (IL-8(1-77) or monocyte derived IL-8 (IL-8(1-72) to rabbits produced a concentration-dependent increase in plasma extravasation and an accumulation of polymorphonuclear leukocytes (PMNs) when measured over a 3 h time period. When plasma extravasation and PMN accumulation were measured over a 30 min time period no significant increases in PMN accumulation or plasma extravasation were observed in response to IL-8 alone. However, under these conditions, the addition of prostaglandin E2 (100 pmol) produced a significant potentiation of IL-8-induced plasma extravasation. There was no significant difference between the biological activities of IL-8(1-77) and IL-8(1-72). 2. Plasma extravasation and PMN accumulation induced by IL-8 were inhibited in rabbits pretreated with the monoclonal antibody designated IB4 (1 mg kg-1, i.v.) directed against the common beta chain (CD18) of the leukocyte integrins. 3. The intra-articular administration to rabbits of IL-8(1-77) (1 nmol) resulted 24 h later in the appearance of a mixed population of leukocytes (PMNs and mononuclear cells) in synovial lavage fluid. Biochemical analyses revealed the presence of an increased level of sulphated proteoglycans (sPG) and of the metalloproteinase stromelysin. Pretreatment of rabbits with IB4 (3 mg kg-1, i.v.) inhibited the accumulation of PMNs but had no effect on the mononuclear infiltrate nor on the levels of sPG or stromelysin. 4. The intradermal or intra-articular injection of E. coli-derived endotoxin induced similar inflammatory changes to those observed with IL-8.The possibility that the biological activities of IL-8 were attributable to minor contamination with endotoxin is unlikely for two reasons. Firstly, biological effects of endotoxin were observed at levels greater than that contained in the IL-8 preparation. Secondly,reduction of the endotoxin content of the IL-8 preparation by a factor of 10 did not produce a concomitant reduction in the observed biological activity of the IL-8.

CAMP-dependent induction of fatty acid cyclooxygenase mRNA in mouse osteoblastic cells (MC3T3-E1)

In an osteoblastic cell line, MC3T3-E1, cloned from mouse calvaria, epinephrine stimulated the production of prostaglandin E2 as an essentially sole arachidonate metabolite (Kusaka, M., Oshima, T., Yokota, K., Yamamoto, S., and Kumegawa, M. (1988) Biochim. Biophys. Acta. 972, 339-346). Western and Northern blot analyses showed increases in the enzyme protein and mRNA of fatty acid cyclooxygenase in the epinephrine-treated cells. A rapid cAMP production caused by epinephrine was followed by increases in the activity and mRNA of cyclooxygenase. Both dibutyryl cAMP and 8-bromo-cAMP also increased the level of the cyclooxygenase activity and mRNA. These results suggest that cAMP produced by beta-adrenergic stimulation was responsible for the increased cyclooxygenase mRNA level leading to induction of the cyclooxygenase enzyme. Furthermore, the addition of prostaglandin E2 (the final arachidonate metabolite in the MC3T3-E1 cells) brought about a rapid synthesis of intracellular cAMP followed by increases in the enzyme protein and mRNA of cyclooxygenase.

Cumulative dose-response curves for bethanechol-induced electrogenic secretion in rat jejunum in vitro: is tachyphylaxis a significant factor?

Electrogenic secretion was monitored as the short-circuit current (Isc) by an automatic voltage clamp across the isolated rat jejunum incubated in vitro. Responses to consecutive additions of bethanechol (1 mM) to the serosal surface showed tachyphylaxis. Addition of prostaglandin E2, however, after a single dose of 1 mM-bethanechol, induced an undiminished Isc response indicating that the tachyphylaxis was homologous. Dose-response curves for serosal applications of bethanechol obtained by serial-cumulative addition did not show tachyphylaxis when compared with those from a non-cumulative technique. The method of automatically recording the intestinal Isc with serial-cumulative addition of a serosal secretagogue gives dose-response curves free from tachyphylaxis.

Neural control of vascular permeability: interactions between primary afferents, mast cells, and sympathetic efferents

1. This study addressed the contribution of primary afferents, mast cells, and sympathetic efferents to the control of vascular permeability in synovial joints. Extravasation of Evans blue dye into the synovial space was measured by perfusion of the knee joint in the adult rat. Plasma extravasation (PE) was evoked by pharmacologic activation of either unmyelinated primary afferents, mast cells, or sympathetic postganglionic nerve (SPGN) terminals with acute injection of either capsaicin, compound 48/80, or 6-hydroxydopamine (6-OHDA), respectively. In otherwise untreated control rats, acute infusion of capsaicin or compound 48/80 produced a brief increase in vascular permeability; infusion of 6-OHDA produced a larger and more prolonged increase. 2. To evaluate the contribution of an interaction of different cellular elements in the joint to PE, we repeated these experiments in rats pretreated with capsaicin, compound 48/80, or 6-OHDA; administered quercetin; or surgically sympathectomized by excision of the lumbar sympathetic chain. Eliminating unmyelinated afferent nerve terminals by neonatal treatment with capsaicin only reduced the increase in PE produced by acute infusion of capsaicin. Degranulating mast cells by pretreatment with compound 48/80, or preventing the degranulation of mast cells by treatment with quercetin, reduced the increase in PE evoked by infusion of either capsaicin or compound 48/80. Finally, sympathectomy, produced by excision of the lumbar sympathetic chain or by pretreatment with 6-OHDA, significantly reduced PE elicited by acute infusion of capsaicin, compound 48/80, or 6-OHDA. 3. Neither infusing substances normally localized to sympathetic efferents nor inducing changes in blood pressure could mimic the profound increase in PE evoked by activation of sympathetic postganglionic neurons with acute infusion of 6-OHDA. Thus norepinephrine produced a significant decrease in PE, adenosine triphosphate produced only a brief increase, neuropeptide Y had no effect, and manipulating blood pressure (either up or down) had no effect on either base-line or 6-OHDA-induced PE. 4. Indomethacin treatment significantly reduced the increase in PE produced by 6-OHDA. This effect of indomethacin was reversed by the addition of prostaglandin E2 (PGE2) to the 6-OHDA in the perfusion fluid. This finding implicates prostaglandins (i.e., cyclooxygenase products of arachidonic acid metabolism) in SPGN-dependent generation of PE.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune parameters of untrained or exercise-trained rats after exhaustive exercise.

The effect of a single exhaustive swimming exercise bout on immune competence of untrained or exercise-trained female Wistar rats was compared with the competence of control sedentary rats. After the exhaustive exercise bout, the blastogenic response to concanavalin A by spleen cells of untrained rats was extensively suppressed, whereas the response of the trained rats was only marginally suppressed. The suppressed immune competence of the untrained rats after the exhaustive exercise was associated with an increase in immune-suppressive activity of splenic lymphocytes. The macrophages of the untrained rats and of the control sedentary rats were slightly immune suppressive to normal spleen cells through a prostaglandin-dependent mechanism. The addition of prostaglandin E2 (PGE2) to the blastogenesis cultures revealed that the spleen cells of untrained rats were unusually sensitive to the suppressive effects of PGE2. In contrast to the untrained rats, the marginal level of immune suppression in trained rats after the exhaustive exercise was associated with a lesser degree of lymphocyte-suppressive activity, an immune stimulatory activity by the splenic macrophages, and an insensitivity of the splenic lymphocytes to the suppressive effects of PGE2.

Interleukin-1 stimulation of phospholipase activity in rat synovial fibroblasts. Possible regulation by cyclooxygenase products.

Tritiated arachidonic acid (3H-AA)-labeled rat synovial fibroblasts stimulated with human recombinant interleukin-1 beta (rIL-1 beta) released incorporated radiolabel in a time-dependent and dose-dependent manner, with labeled prostaglandins representing 29% of the released radiolabel. Treatment of the cells with dibutyryl cAMP or prostaglandin E2 enhanced both spontaneous and rIL-1 beta-induced 3H-AA release; treatment with indomethacin or naproxen inhibited the response. The effects of these cyclooxygenase inhibitors on 3H-AA release were not reversed by the addition of prostaglandin E2. The activities of phospholipase A, phospholipase C, and diglyceride lipase were detected in the homogenates of rat synovial fibroblasts. Pretreatment of synovial cells with rIL-1 beta resulted in a threefold stimulation of phospholipase A activity and a slight increase in phospholipase C activity in cell homogenates. These data show that rIL-1 beta stimulates phospholipase activities in rat synovial fibroblasts and that at least one of these activities may be regulated by either prostaglandins or cAMP.

Regulation of human natural killer (NK) cell function: induction of killing of an NK-resistant renal carcinoma cell line.

Natural killer (NK)-like activity against a renal carcinoma cell line, Cur, was assessed. There was no spontaneous killing of Cur cells by human peripheral blood mononuclear cells in 4-hr assays. Cur killing was observed in 18-hr assays, but the magnitude of killing was variable and always markedly less than that against K562. Cur killing was mediated by a nonadherent, nonphagocytic lymphocyte, the activity of which could be modulated both positively and negatively by monocytes or their products. Preincubation of effectors with monocyte supernatant, interleukin 1 (IL-1), alpha-interferon (alpha IFN), or interleukin 2 (IL-2) greatly increased the magnitude of Cur killing and accelerated the kinetics of lysis. The addition of prostaglandin E2 (PGE2) during in vitro activation of NK by IL-2 profoundly inhibited subsequent Cur lysis, whereas only minimal inhibition of K562 lysis was noted. However, following activation with IL-2, lysis of Cur targets was less sensitive to the inhibitory effects of PGE2. Removal of Leu 11b(+), OKM1(+), or L-leucyl-leucine methyl ester-sensitive cells markedly decreased both Cur and K562 lysis. Moreover, CD16(+) cells purified with the fluorescence-activated cell sorter were found to mediate Cur killing. Whereas Cur and K562 lysis is mediated by phenotypically similar effector cells, the present studies demonstrate that the cytotoxic functions defined by the ability to lyse these two targets differ in response to a variety of immunoregulatory stimuli.

Cyclic adenosine 3',5'-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins.

The role and mechanism of action of cyclic adenosine 3',5'-monophosphate (cAMP) in the ovulatory process was investigated by using the in vitro-perfused rat ovary model. Ovaries of pregnant mare's serum gonadotropin (PMSG, 20 IU)-primed rats were perfused for 21 h beginning in the morning of induced proestrus. In vitro stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) resulted in 2.4 +/- 0.7 ovulations per treated ovary. Ovulations could also be induced by the addition of forskolin (30 microM) or dibutyryl cAMP (dbcAMP, 1 mM) with isobutylmethylxanthine (IBMX, 0.2 mM), with 11.8 +/- 1.9 and 18.6 +/- 4.4 ovulations per treated ovary, respectively. Indomethacin (5 micrograms/ml) significantly decreased the number of ovulations in the forskolin and dbcAMP + IBMX groups. The addition of prostaglandin E2 (PGE2; 1 micrograms/ml three times during the perfusion) to the forskolin + indomethacin group reversed the inhibition of ovulation (21.6 +/- 5.4 ovulations per treated ovary). Ovarian PGE tissue levels were significantly higher 10 h after stimulation with either LH, forskolin, or dbcAMP + IBMX compared to the unstimulated control group. Ovulated oocytes in the LH and forskolin groups resumed meiosis but oocytes in the dbcAMP + IBMX groups remained immature. This study shows that an increase in ovarian cAMP, even if not induced by LH, is sufficient to cause ovulation of preovulatory rat follicles, supporting the involvement of cAMP in the normal ovulatory process of the PMSG-treated rat. Furthermore, prostaglandin involvement in cAMP-induced ovulations is demonstrated.