Interaction of prostaglandins with blood plasma proteins. I. Binding of prostaglandin E 2 to human plasma proteins and its effect on the physiological activity of prostaglandin E 2 in vitro and in vivo

The binding of (PGE2) prostaglandin E2 to human plasma proteins was investigated by DEAE-Sephadex column chromatography and acrylamide gel electrophoresis, and quantitatively assessed by equilibrium dialysis. PGE2 added to human plasma in vitro was found to become mainly bound to plasma albumin. This binding was also demonstrated by adding PGE2 to human serum albumin solutions. The binding of PGE2 to human serum albumin inhibits the contraction-producing effect of PGE2 on the isolated gerbil colon in vitro. The depressor effect of PGE2 on the rat blood pressure was used to assess the in vivo effect of PGE2 albumin interaction. The blood pressure lowering activities of free and albumin-bound PGE2 were found to be the same when administered either intravenously or intraarterially. The significance of these observations with regard to estimation of PG concentration in whole blood or plasma, and their possible effects on PG metabolism is discussed.     

Growth stimulation by PGE2 and EGF activates cyclic AMP-dependent and -independent pathways in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells from virgin Balb/c mice were isolated by collagenase digestion and cultured within collagen gels in serum-free basal medium containing insulin (10 micrograms/ml). Previous work has shown that linoleate or its metabolite, prostaglandin E2 (PGE2), stimulate the growth of these cells only in the presence of a growth stimulant such as epidermal growth factor (EGF). Since PGE2 can stimulate cyclic AMP (cAMP) production, the role of cAMP in linoleate and EGF-stimulated growth was examined. The cAMP phosphodiesterase inhibitor, IBMX (0.1 mM), was found to augment growth when cells were cultured in the presence of both EGF and linoleate or PGE2, but not either factor alone. These results indicated that EGF does not stimulate proliferation via cyclic AMP mediated events but could synergize with cAMP events if cAMP levels were elevated by PGE2. When assayed in cells plated on top of collagen-coated culture dishes, cellular cyclic AMP levels were stimulated by PGE2, but only marginally by EGF. Although the stimulation of endogenous cAMP by PGE2 and IBMX was insufficient to stimulate growth in the absence of EGF, exogenous dibutyryl-cAMP (greater than 100 micrograms/ml) was able to do so showing that a sustained, and high level of cAMP (greater than 100 micrograms/ml) could stimulate growth in insulin-containing basal medium. EGF was capable of enhancing the cellular sensitivity to dibutyryl-cAMP but the converse was not observed. cAMP stimulation of growth was dependent upon a superphysiological concentration of insulin (10 micrograms/ml) or a physiological concentration of somatomedin-C. These results indicate that the proliferation of mouse mammary epithelial cells can be stimulated separately or in synergism by cAMP-dependent or -independent events.     

Involvement of tyrosine kinases on cyclooxygenase expression and prostaglandin E2 production in human gingival fibroblasts stimulated with interleukin-1beta and epidermal growth factor

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tyrosine kinase on prostaglandin E2 (PGE2) production in human gingival fibroblasts stimulated by interleukin-1beta (IL-1beta) and/or epidermal growth factor (EGF). The cytokine IL-1beta and to a lesser extent EGF, enhanced COX-2 mRNA levels in gingival fibroblasts. Simultaneous treatment with EGF and IL-1beta resulted in enhanced COX-2 mRNA levels accompanied by a synergistic stimulation of PGE2 biosynthesis compared to the cells treated with IL-1beta or EGF alone. Neither IL-1beta EGF nor the combination of IL-1beta and EGF enhanced COX-1 mRNA levels in gingival fibroblasts. The tyrosine kinase inhibitors, Herbimycin A and PD 153035 hydrochloride, reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta or the combination of IL-1beta and EGF whereas COX-1 mRNA levels were not affected. Furthermore, the COX-2 specific inhibitor, NS-398, abolished the PGE2 production induced by IL-1beta, EGF, or the combination. These results indicate that the synergy between IL-1beta and EGF on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in gingival fibroblasts.     

Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.     

Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice

The effects of zinc supplementation (20 mM ZnCl₂ from the drinking water for eight weeks) on plasma glucose and insulin levels, as well as its in vitro effect on lipogenesis and lipolysis in adipocytes were studied in genetically obese (ob/ob) mice and their lean controls (+/?). Zinc supplementation reduced the fasting plasma glucose levels in both obese and lean mice by 21 and 25%, respectively (p < 0.05). Fasting plasma insulin levels were significantly decreased by 42% in obese mice after zinc treatment. In obese mice, zinc supplementation also attenuated the glycemic response by 34% after the glucose load. The insulin-like effect of zinc on lipogenesis in adipocytes was significantly increased by 80% in lean mice. However, the increment of 74% on lipogenesis in obese mice was observed only when the zinc plus insulin treatment was given. This study reveals that zinc supplementation alleviated the hyperglycemia of ob/ob mice, which may be related to its effect on the enhancement of insulin activity.     

Fatty acid and prostaglandin metabolism in children with diabetes mellitus. II. The effect of evening primrose oil supplementation on serum fatty acid and plasma prostaglandin levels

Our previous study demonstrated that levels of dihomo-gamma-linolenic acid (DGLA) and arachidonic acid in serum total lipids decreased in association with increased plasma levels of prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha) in patients with insulin-dependent diabetes mellitus. In this study, 11 children with insulin-dependent diabetes mellitus completed a double-blind, placebo-controlled study to assess the effect of dietary supplementation with gamma-linolenic acid (GLA) on serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. GLA was given as the seed oil from the evening primrose (EPO) and all patients received either EPO capsules (containing 45 mg of GLA and 360 mg of linoleic acid) or indistinguishable placebo capsules for 8 months. Initially patients took 2 capsules daily for 4 months then 4 capsules daily for a further 4 months. All patients were assessed at the start of the study, after 4 months and at the end of the study, by measuring serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. After administration of 4 capsules daily the DGLA levels increased and PGE2 levels decreased significantly (p less than 0.01) in the EPO compared with the placebo group. Neither fatty acid nor PGE2 and PGF2 alpha levels were altered by administration of 2 EPO capsules daily. This suggests that the altered essential fatty acid and PG metabolism in diabetes may be reversed by direct GLA supplementation.     

The epidermal growth factor receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable guanine nucleotide-binding regulatory protein in cultured rat inner medullary collecting tubule cells

Studies were performed to examine a potential role for a guanine nucleotide-binding protein in epidermal growth factor (EGF)-stimulated phospholipase A2 (PLA2) activity. EGF increased prostaglandin E2 (PGE2) production in intact or saponin-permeabilized rat inner medullary collecting tubule (RIMCT) cells. Incubation of permeabilized cells with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) enhanced and with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) inhibited the response to EGF. GDP beta S had no effect on ionomycin-stimulated PGE2 production. Exposure of intact cells to 25 mM NaF + 10 microM AlCl3 enhanced both basal and EGF-stimulated PGE2 production. Pertussis toxin ADP-ribosylated a 41-kDa protein in RIMCT cell membranes. Pretreatment of cells with pertussis toxin (100 ng/ml for 16 h) eliminated the response to EGF in intact cells and the response to EGF + GTP gamma S in permeabilized cells. Pertussis toxin had no effect on the response to ionomycin. The effect of pertussis toxin was not due to alterations in cAMP as cellular cAMP levels were unaffected by pertussis toxin both in the basal state and in the presence of EGF. PGE2 production in response to EGF was not transduced by a G protein coupled to phospholipase C (PLC) as neomycin, which inhibited PLC, did not decrease EGF-stimulated PGE2 production. Also, PGE2 production was not increased by inositol trisphosphate and did not require the presence of extracellular Ca2+. In contrast to EGF-stimulated PLC activity, stimulation of PLA2 by EGF was not susceptible to inhibition by phorbol 12-myristate 13-acetate. These results clearly demonstrate the existence of a PLA2-specific pertussis toxin-inhibitable guanine nucleotide-binding protein coupled to the EGF receptor in RIMCT cells.     

Differentiation status-dependent regulation of cyclooxygenase-2 expression and prostaglandin E2 production by epidermal growth factor via mitogen-activated protein kinase in articular chondrocytes

Although large amounts of epidermal growth factor (EGF) are found in the synovial fluids of arthritic cartilage, the role of EGF in arthritis is not clearly understood. This study investigated the effect of EGF on differentiation and on inflammatory responses such as cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in articular chondrocytes. EGF caused a loss of differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen expression and proteoglycan synthesis. EGF also induced COX-2 expression and PGE(2) production. EGF-induced dedifferentiation was caused by EGF receptor-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) but not p38 kinase, whereas the activation of both ERK1/2 and p38 kinase was necessary for COX-2 expression and PGE(2) production. Neither the inhibition of COX-2 expression and PGE(2) production nor the addition of exogenous PGE(2) affected EGF-induced dedifferentiation. However, COX-2 expression and PGE(2) production were significantly enhanced in chondrocytes that were dedifferentiated by serial subculture, and EGF also potentiated COX-2 expression and PGE(2) production, although these cells were less sensitive to EGF. Dedifferentiation-induced COX-2 expression and PGE(2) production were mediated by ERK1/2 and p38 kinase signaling. Our results indicate that EGF in articular chondrocytes stimulates COX-2 expression and PGE(2) production via ERK and p38 kinase signaling in association with differentiation status.     

The effect of sialoadenectomy on gastric mucosal integrity in the rat: roles of epidermal growth factor and prostaglandin E2

Removal of the salivary glands (SALX) in rats has been shown to increase the susceptibility of gastric mucosa to ulcerogens. In the present study, we have investigated the role of specific salivary glands in this response. In addition, we have examined whether a functional link exists between the salivary glands, epidermal growth factor (EGF), and prostaglandin E2 (PGE2) by determining whether SALX decreases the responsiveness of the mucosa to the protective actions of either of both of these agents. Removal of the parotid salivary glands did not significantly increase ulceration in response to intragastric administration of 100% w/v ethanol. Animals were examined 60 min after ethanol administration. Removal of the submandibular-sublingual gland complexes was associated with a significant increase in the area of mucosal damage and a decrease in gastric pit depth in ethanol-treated animals when compared with sham-operated control rats. Furthermore, in both SALX and control animals, exogenous PGE2 and EGF resulted in a dose-dependent reduction in both groups of animals, although the protective effects of PGE2 and EGF were attenuated in SALX rats. PGE2 and EGF administered in combination resulted in the same degree of protection in both SALX and control rats. Sialoadenectomy resulted in a reduction in mucosal PGE2 synthesis. EGF administration did not consistently increase mucosal PGE2 synthesis. Conversely, sialoadenectomy did not reduce mucosal levels of EGF nor did exogenous PGE2 consistently increase salivary or mucosal content of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential PGE2 and cAMP production by allogeneic and syngeneic pregnant mice uteri.

Prostaglandin E2 (PGE2) and cAMP production in allogeneic and syngeneic pregnant mice uteri, were measured in relation to the ratio of plasma estrogen/progesterone levels. PGE2 generation by allopregnant uteri varied with the days of pregnancy. The increment of the prostanoid coincided with the increase in plasma estrogen concentration, whereas the decrement of its production was in parallel with the increment of plasma progesterone. The syngeneic pregnant uterus was unable to increase the release of PGE2 above basal values during the whole pregnancy. The rise of PGE2 production by the allogeneic pregnant uterus was correlated with an increase in cAMP levels. It is proposed that the pregnant mouse uterus increases its ability to release PGE2 in response to an ovarian steroids.     

Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE2) which has a longer half-life in blood. We examined the effects of PGE2 infusion and alterations in lipolysis in vivo, and of clotting, prolonged storage and hemolysis in vitro, on KH2-PGE2 immunoreactivity in unextracted human plasma and serum samples. Indeed KH2-PGE2 levels rose several hundred fold during infusions of PGE2 at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH2-PGE2 levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH2-PGE2 levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH2-PGE2 levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH2-PGE2 measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores in vivo or from disrupted membrane phospholipids in vitro. Unextracted plasma appears to be unsatisfactory for use in this RIA.     

Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.     

Bovine milk-derived α-lactalbumin inhibits colon inflammation and carcinogenesis in azoxymethane and dextran sodium sulfate-treated mice

Cyclooxygenase-2 is expressed early in colon carcinogenesis and plays crucial role in the progress of the disease. Recently, we found that α-lactalbumin had anti-inflammatory activity by inhibiting cyclooxygenase-2. In experiment 1, we investigated the effects of α-lactalbumin on the colon carcinogenesis initiated with azoxymethane (AOM) followed by promotion with dextran sodium sulfate (DSS) in mice. Dietary treatment with α-lactalbumin decreased fecal occult blood score at 3?days after DSS intake. α-Lactalbumin also decreased the colon tumor at week 9. In experiment 2, AOM-treated mice were sacrificed at 7?days after DSS intake. The plasma and colon prostaglandin E2 (PGE2) levels in AOM/DSS-treated mice were higher than those in the DSS-treated mice without initiation by AOM. α-Lactalbumin decreased PGE2 in both plasma and colon. These results suggest that α-lactalbumin effectively inhibited colon carcinogenesis, and the inhibition may be due to the decreased PGE2 by inhibiting cyclooxygenase-2 at cancer promotion stages.     

丁香苷抗炎镇痛作用及部分机制研究

研究丁香苷抗炎镇痛作用及部分机制。以阿司匹林作阳性对照药,观察丁香苷对二甲苯致小鼠耳廓肿胀、醋酸致小鼠毛细血管通透性增加、角叉菜胶致大鼠足趾肿胀、棉球致大鼠肉芽肿的抗炎作用;对小鼠热板试验、醋酸扭体试验的镇痛作用;同时测定角叉菜胶致大鼠炎足炎性渗出物中的PGE2、MDA和血清中的NO、SOD,初步探讨丁香苷抗炎镇痛的部分机制。结果表明,丁香苷对急慢性炎症反应有明显抑制作用,能明显降低角叉菜胶致炎足炎性渗出物中PGE2、MDA和血清中NO含量,明显增加血清中SOD的活性。因此,丁香苷具有较强的抗炎镇痛作用,其机制可能与抑制PGE2、NO等炎症介质生成、增强自由基清除能力有关。     

Protective role of adiponectin against ethanol-induced gastric injury in mice

Adiponectin is an anti-inflammatory molecule released from adipocytes, and serum adiponectin concentrations are reduced in obesity. We previously reported that gastric erosion occurs in association with obesity and low serum adiponectin levels. In the present study, we examined adiponectin-knockout (APN-KO) mice to elucidate the role of adiponectin in gastric mucosal injury. Gastric injury was induced by oral administration of ethanol in wild-type (WT) and APN-KO mice. Ethanol treatment induced severe gastric injury in APN-KO mice compared with WT mice. In APN-KO mice, increased apoptotic cells and decreased expression of prostaglandin E(2) (PGE(2)) were detected in the injured stomach. We next assessed the effect of adiponectin on the cellular response to ethanol treatment and wound repair in rat gastric mucosal cells (RGM1). Adiponectin induced the expression of PGE(2) and cyclooxygenase 2 (COX-2) in ethanol-treated RGM1 cells. RGM1 cells exhibited efficient wound repair accompanied by increased PGE(2) expression in the presence of adiponectin. Coadministration of adiponectin with celecoxib, a COX-2 inhibitor, inhibited efficient wound repair. These findings indicate that adiponectin has a protective role against ethanol-induced gastric mucosal injury in mice. This effect may be partially mediated by the efficient wound repair of epithelial cells via increased PGE(2) expression.     

Effect of pertussis toxin on the inhibition of secretory activity by prostaglandin E2, somatostatin, epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate in parietal cells from rat stomach

Rat parietal cells were incubated for 2 h with pertussis toxin (100 ng/ml) which ADP-ribosylates and inactivates guanine nucleotide regulatory proteins (G proteins) of the 'Gi-like' family. The effect of this pretreatment on the action of inhibitors of parietal cell acid secretion was investigated by using the accumulation of the weak base aminopyrine as an index of secretory activity. The inhibitory actions of near maximally effective concentrations of prostaglandin E2 (PGE2), somatostatin and epidermal growth factor (EGF) on histamine-stimulated aminopyrine accumulation were reduced by 83%, 72% and 70%, respectively, by preincubation with pertussis toxin. By contrast, the inhibitory action of a near maximally effective concentration of 12-O-tetradecanoylphorbol 13-acetate on histamine-stimulated aminopyrine accumulation was reduced by only 12%. It is concluded that G-proteins are involved in the inhibitory actions of PGE2, somatostatin and EGF on parietal cells. However, since the inhibitory actions of PGE2 and EGF can be distinguished by the blockade of the action of EGF, but not that of PGE2, by 3-isobutyl-1-methylxanthine, it is possible that PGE2 and EGF either activate the same G-protein in different ways or work through different G-proteins.     

Positive feedback loop between prostaglandin E2 and EGF-like factors is essential for sustainable activation of MAPK3/1 in cumulus cells during in vitro maturation of porcine cumulus oocyte complexes

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.     

Effects of CL 115,347, (+/-)-15-deoxy-16-hydroxy-16-vinyl-PGE2 methyl ester, on cardiovascular responses and plasma catecholamines in pithed stroke-prone spontaneously hypertensive rats during sympathetic stimulation

The effect of CL 115,347, a topically active antihypertensive PGE2 analog, and PGE2 on changes in blood pressure (BP), heart rate (HR) response and plasma epinephrine (E) and norepinephrine (NE) levels induced by stimulation of the sympathetic spinal cord outflow were studied in pithed stroke-prone spontaneously hypertensive rats (SHRSP). Surgical pithing significantly reduced plasma E but not NE levels suggesting that the sympathoadrenal medullary system differentially affects E and NE release. Sympathetic stimulation of the spinal cord of pithed SHRSP increased HR, BP, plasma E and NE levels. Topically applied CL 115,347 (0.001-0.2 mg/kg) dose-dependently decreased BP, while intravenously infused PGE2 (30 micrograms/kg/min) did not alter BP except for a brief initial drop. Topical application of CL 115,347 (0.1 mg/kg) also inhibited BP responses to sympathetic stimulation without effects on HR or plasma E or NE levels. Intravenous infusion of PGE2 (30 micrograms/kg/min) inhibited both BP and HR responses to spinal cord stimulation but did not alter plasma catecholamine levels. These studies in SHRSP suggest that CL 115,347 and PGE2 modulate cardiovascular responses mainly via postjunctional effects, but act differently on the cardiovascular elements, viz. CL 115,347 acts primarily on blood vessels while PGE2 acts on blood vessels and heart.     

PGE1-independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum-free medium

Prostaglandin E1 (PGE1), a component in the hormone-supplemented, serum-free medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1-independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MDCK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K-1 (9.0 pmol/mg protein) than in Medium K-1 minus PGE1. Furthermore, PGE1-independent clone 1 had higher intracellular cyclic AMP levels in Medium K-1 minus PGE1 than normal MDCK cells in Medium K-1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1-independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1-independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1-independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP-mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K-1. Thus, EGF and PGE1 could possibly affect similar growth-related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1-independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1-independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.